U.S. patent application number 17/045952 was filed with the patent office on 2021-06-03 for certain pladienolide compounds and methods of use.
This patent application is currently assigned to EISAI R&D MANAGEMENT CO., LTD.. The applicant listed for this patent is EISAI R&D MANAGEMENT CO., LTD.. Invention is credited to Kenzo ARAI, Andrew COOK, Shelby ELLERY, Atsushi ENDO, Nicholas C. GEARHART, Baudouin GERARD, Ming-Hong HAO, Regina Mikie KANADA SONOBE, Gregg F. KEANEY, Kazunobu KIRA, Yoshihiko KOTAKE, Xiang LIU, Jason T. LOWE, Touping LUO, Lisa A. MARCAURELLE, Masayuki MIYANO, Norio MURAI, Satoshi NAGAO, Marta NEVALAINEN, Morgan Welzel O'SHEA, James PALACINO, Sudeep PRAJAPATI, Dominic REYNOLDS, Parcharee TIVITMAHAISOON, John WANG, Guo Zhu ZHENG.
Application Number | 20210163456 17/045952 |
Document ID | / |
Family ID | 1000005400395 |
Filed Date | 2021-06-03 |
United States Patent
Application |
20210163456 |
Kind Code |
A1 |
KEANEY; Gregg F. ; et
al. |
June 3, 2021 |
CERTAIN PLADIENOLIDE COMPOUNDS AND METHODS OF USE
Abstract
The present disclosure provides novel pladienolide compounds,
pharmaceutical compositions containing such compounds, and methods
for using the compounds as therapeutic agents. These compounds may
be useful in the treatment of cancers, particularly cancers in
which agents that target the spliceosome and mutations therein are
known to be useful. Also provided herein are methods of treating
cancers by administering at least one compound disclosed herein and
at least one additional therapy.
Inventors: |
KEANEY; Gregg F.;
(Lexington, MA) ; WANG; John; (Andover, MA)
; GERARD; Baudouin; (Belmont, MA) ; ARAI;
Kenzo; (Ibaraki, JP) ; LIU; Xiang;
(Winchester, MA) ; ZHENG; Guo Zhu; (Lexington,
MA) ; KIRA; Kazunobu; (Ibaraki, JP) ;
MARCAURELLE; Lisa A.; (Arlington, MA) ; NEVALAINEN;
Marta; (Weymouth, MA) ; HAO; Ming-Hong;
(Quincy, MA) ; O'SHEA; Morgan Welzel; (Arlington,
MA) ; TIVITMAHAISOON; Parcharee; (Boston, MA)
; PRAJAPATI; Sudeep; (Somerville, MA) ; LUO;
Touping; (Newton, MA) ; GEARHART; Nicholas C.;
(Durango, CO) ; LOWE; Jason T.; (East Bridgewater,
MA) ; KOTAKE; Yoshihiko; (Ibaraki, JP) ;
NAGAO; Satoshi; (Ibaraki, JP) ; KANADA SONOBE; Regina
Mikie; (Ibaraki, JP) ; MIYANO; Masayuki;
(Ibaraki, JP) ; MURAI; Norio; (Ibaraki, JP)
; COOK; Andrew; (Stow, MA) ; ELLERY; Shelby;
(Boston, MA) ; ENDO; Atsushi; (Andover, MA)
; PALACINO; James; (Wellesley, MA) ; REYNOLDS;
Dominic; (Stoneham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
EISAI R&D MANAGEMENT CO., LTD. |
Tokyo |
|
JP |
|
|
Assignee: |
EISAI R&D MANAGEMENT CO.,
LTD.
Tokyo
JP
|
Family ID: |
1000005400395 |
Appl. No.: |
17/045952 |
Filed: |
April 8, 2019 |
PCT Filed: |
April 8, 2019 |
PCT NO: |
PCT/US2019/026313 |
371 Date: |
October 7, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62814843 |
Mar 6, 2019 |
|
|
|
62814838 |
Mar 6, 2019 |
|
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|
62679653 |
Jun 1, 2018 |
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62655021 |
Apr 9, 2018 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07D 417/12 20130101;
A61P 35/00 20180101; C07D 487/10 20130101; C07D 405/14 20130101;
C07D 491/107 20130101; A61K 45/06 20130101; C07D 487/08 20130101;
C07D 407/06 20130101; C07D 313/00 20130101; C07D 405/12 20130101;
C07D 413/12 20130101 |
International
Class: |
C07D 407/06 20060101
C07D407/06; C07D 405/14 20060101 C07D405/14; A61P 35/00 20060101
A61P035/00; C07D 313/00 20060101 C07D313/00; C07D 491/107 20060101
C07D491/107; C07D 487/08 20060101 C07D487/08; C07D 487/10 20060101
C07D487/10; A61K 45/06 20060101 A61K045/06; C07D 405/12 20060101
C07D405/12; C07D 417/12 20060101 C07D417/12; C07D 413/12 20060101
C07D413/12 |
Claims
1. A compound chosen from compounds of Formula I: ##STR00607## and
pharmaceutically acceptable salts thereof, wherein: n is chosen
from 0, 1, 2 or 3; R.sup.1 is chosen from C.sub.1-C.sub.6 alkyl
groups, C.sub.3-C.sub.8 cycloalkyl groups, --NR.sup.9R.sup.10,
##STR00608## groups, ##STR00609## groups, ##STR00610## groups,
##STR00611## groups, ##STR00612## groups, ##STR00613## groups,
##STR00614## groups, and ##STR00615## groups; R.sup.9 is chosen
from hydrogen, --NR.sup.11R.sup.12 groups, C.sub.1-C.sub.6 alkyl
groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H groups,
C.sub.3-C.sub.8 cycloalkyl groups, and C.sub.3-C.sub.8 heterocyclyl
groups, wherein the --NR.sup.11R.sup.12 groups, C.sub.1-C.sub.6
alkyl groups, C.sub.3-C.sub.8 cycloalkyl groups, and
C.sub.3-C.sub.8 heterocyclyl groups may be unsubstituted or
substituted from 1-3 times with a group independently chosen from
C.sub.1-C.sub.6 alkyl groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H
groups, hydroxy, halogen groups, and C.sub.1-C.sub.6 alkoxy groups;
R.sup.10 is chosen from hydrogen and C.sub.1-C.sub.6 alkyl groups;
one of either R.sup.2 or R.sup.3 is chosen from hydrogen and
C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, --OR.sup.10, --OC(O)R.sup.10, --OC(O)R.sup.1, and
C.sub.1-C.sub.6 alkyl groups; R.sup.4 is chosen from hydrogen and
hydroxy; R.sup.5 and R.sup.6 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups; R.sub.7 and R.sub.8 are each
independently chosen from hydrogen, hydroxy, C.sub.1-C.sub.6 alkoxy
groups, and C.sub.1-C.sub.6 alkyl groups; and Y is chosen from
phenyl, thiophenyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl,
and pyrazinyl, wherein Y may be unsubstituted or substituted from
1-3 times with groups independently chosen from oxo groups,
C.sub.1-C.sub.6 alkyl groups, C.sub.3-C.sub.5 cycloalkyl groups,
hydroxy C.sub.1-C.sub.6 alkyl groups, C.sub.1-C.sub.6 alkoxy
groups, methoxy C.sub.1-C.sub.6 alkyl groups, --NR.sup.11R.sup.12
groups, ##STR00616## wherein R.sup.11 and R.sup.12 are each
independently chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups.
2. The compound of claim 1, wherein Y is ##STR00617##
3. The compound of claim 1, wherein Y is optionally substituted
phenyl.
4. The compound of claim 1, wherein R.sup.1 is chosen from methyl,
##STR00618## groups, ##STR00619## groups, ##STR00620## groups,
##STR00621## groups, ##STR00622## groups, ##STR00623## groups,
##STR00624## groups, and ##STR00625## groups.
5. A compound chosen from compounds of Formula II: ##STR00626## and
pharmaceutically acceptable salts thereof, wherein: X is chosen
from O, NR' groups, and CH.sub.2, wherein R' is chosen from
hydrogen and C.sub.1-C.sub.6 alkyl groups; R.sup.1 is chosen from
methyl, --NR.sup.11R.sup.12 groups, ##STR00627## groups, and
##STR00628## groups, R.sup.10 is chosen from C.sub.1-C.sub.6 alkyl
groups, C.sub.3-C.sub.8 cycloalkyl groups, and halo C.sub.1-C.sub.6
alkyl groups, wherein the C.sub.3-C.sub.8 cycloalkyl groups may be
unsubstituted or substituted from 1-3 times with a group
independently chosen from C.sub.1-C.sub.6 alkyl groups, hydroxy,
halogen groups, and C.sub.1-C.sub.6 alkoxy groups; R.sup.11 and
R.sup.12 are each independently chosen from C.sub.1-C.sub.6 alkyl
groups; one of either R.sup.2 or R.sup.3 is chosen from hydrogen
and C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, hydroxy and C.sub.1-C.sub.6 alkyl groups; one of either
R.sup.4 or R.sup.5 is hydrogen, and the other is chosen from
hydrogen, hydroxy, and ##STR00629## R.sup.6 and R.sup.7 are each
independently chosen from C.sub.1-C.sub.6 alkyl groups; R.sup.8 and
R.sup.9 are each independently chosen from hydrogen and
C.sub.1-C.sub.6 alkyl groups; or R.sup.8 and R.sup.9 are taken
together to form a cyclopropyl ring; and Y is chosen from
C.sub.1-C.sub.6 alkyl groups, C.sub.3-C.sub.8 cycloalkyl groups,
methoxy, and --NR.sup.13R.sup.14 groups, wherein R.sup.13 and
R.sup.14 are each independently chosen from hydrogen,
C.sub.1-C.sub.6 alkyl groups, and methoxy C.sub.1-C.sub.6 alkyl
groups; or R.sup.13 and R.sup.14 may be taken together with the N
to form a group chosen from ##STR00630## a morpholine, a
piperidine, a thiazolidine, an indole, an indoline, and an
isoindoline ring; wherein Y may be unsubstituted or substituted
from 1-3 times with a group independently chosen from
C.sub.1-C.sub.6 alkyl groups, hydroxy, hydroxy C.sub.1-C.sub.6
alkyl groups, methoxy, methoxy C.sub.1-C.sub.6 alkyl groups, halo,
halo C.sub.1-C.sub.6 alkyl groups, --C(O)NH.sub.2,
--NHCOO--C.sub.1-C.sub.6 alkyl groups, --COOH, ##STR00631## and
--NR.sup.15R.sup.16 groups, wherein R.sup.15 and R.sup.16 are each
independently chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups.
6. A compound chosen from compounds of Formula III: ##STR00632##
and pharmaceutically acceptable salts thereof, wherein: n is chosen
from 0, 1 and 2; m is chosen from 1, 2, and 3; R.sup.1 is chosen
from C.sub.1-C.sub.6 alkyl groups, C.sub.3-C.sub.8 cycloalkyl
groups, --NR.sup.11R.sup.12 groups, ##STR00633## groups,
##STR00634## groups, ##STR00635## groups, ##STR00636## groups,
##STR00637## groups, ##STR00638## groups, ##STR00639## groups,
##STR00640## groups, and ##STR00641## groups, R.sup.11 is chosen
from hydrogen, --NR.sup.16R.sup.17 groups, C.sub.1-C.sub.6 alkyl
groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H groups,
--(C.sub.1-C.sub.6 alkyl)--CO.sub.2R.sup.12 groups,
--(C.sub.1-C.sub.6 alkyl)--NR.sup.16R.sup.17 groups,
C.sub.3-C.sub.8 cycloalkyl groups, and C.sub.3-C.sub.8 heterocyclyl
groups, wherein the --NR.sup.11R.sup.12 groups, C.sub.1-C.sub.6
alkyl groups, C.sub.3-C.sub.8 cycloalkyl groups and C.sub.3-C.sub.8
heterocyclyl groups may be unsubstituted or substituted from 1-3
times with a group independently chosen from C.sub.1-C.sub.6 alkyl
groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H groups, hydroxy,
halogen groups, and C.sub.1-C.sub.6 alkoxy groups; R.sup.12 is
chosen from hydrogen and C.sub.1-C.sub.6 alkyl groups; one of
either R.sup.2 or R.sup.3 is chosen from hydrogen and
C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, --OR.sup.10, --OC(O)R.sup.10, --OC(O)R.sup.1, and
C.sub.1-C.sub.6 alkyl groups; R.sup.4 is hydrogen or hydroxy;
R.sup.5 and R.sup.6 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups; R.sub.7 and R.sub.8 are each
independently chosen from hydrogen, hydroxy, C.sub.1-C.sub.6 alkoxy
groups, and C.sub.1-C.sub.6 alkyl groups; and R.sup.9 and R.sup.10
are each independently chosen from hydrogen, C.sub.1-C.sub.6 alkyl
groups, hydroxy, and C.sub.1-C.sub.6 alkoxy groups; or, one of
R.sup.9 or R.sup.10 is oxo and the other is absent; Z is chosen
from C.sub.1-C.sub.6 alkyl groups, --C(O)--C.sub.1-C.sub.6 alkyl
groups, --OR.sup.13, and --NR.sup.14R.sup.15 groups, wherein
R.sup.13 is chosen from hydrogen, C.sub.1-C.sub.6 alkyl groups, and
--C(O)--C.sub.1-C.sub.6 alkyl groups, wherein R.sup.14 and R.sup.15
are each independently chosen from hydrogen, C.sub.1-C.sub.6 alkyl
groups, and methoxy C.sub.1-C.sub.6 alkyl groups; or R.sup.14 and
R.sup.15 may be taken together with the N to form a group chosen
from ##STR00642## a morpholine, a piperidine, a thiazolidine, an
indole, an indoline, and an isoindoline ring; wherein Z may be
unsubstituted or substituted from 1-3 times with a group
independently chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.5 cycloalkyl groups, hydroxy C.sub.1-C.sub.6 alkyl
groups, C.sub.1-C.sub.6 alkoxy groups, methoxy C.sub.1-C.sub.6
alkyl groups, --NR.sup.16R.sup.17 groups, ##STR00643## wherein
R.sup.16 and R.sup.17 are each independently chosen from hydrogen
and C.sub.1-C.sub.6 alkyl groups.
7. The compound of claim 6, wherein R.sup.1 is chosen from methyl,
##STR00644## groups, ##STR00645## groups, ##STR00646## groups,
##STR00647## groups, ##STR00648## groups, ##STR00649## groups,
##STR00650## groups, ##STR00651## groups, and ##STR00652##
groups.
8. A compound chosen from:
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
9-oxo-9-pyrrolidin-1-ylnona-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-y-
l] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-[[(2R,3R)-3-hydroxyp-
entan-2-yl]carbamoyloxy]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo--
1-oxacyclododec-4-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-(propylcarbamoyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-y-
l] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-[methyl(propyl)carbamoyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec--
4-en-6-yl] acetate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
pyrrolidine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-[methyl(propyl)carbamoyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec--
4-en-6-yl] 4-cycloheptyl-4-oxidopiperazin-4-ium-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(dimethylcarbamoyloxy)-6-methylhept-
a-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-
-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-(diethylcarbamoyloxy)-6-methylhepta-
-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-[methyl(propan-2-yl)carbamoyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclod-
odec-4-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-[butyl(methyl)carbamoyl]oxy-6-methy-
lhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-[butan-2-yl(methyl)carbamoyl]oxy-6--
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclodo-
dec-4-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-carbamoyloxy-6-methylhepta-2,4-dien-
-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3-
,7-dimethyl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(2R)-2-(methoxymethyl)pyrrolidine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-[2-methoxyethyl(meth-
yl)carbamoyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyc-
lododec-4-en-6-yl] acetate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
azetidine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(2S)-2-methylpyrrolidine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(2S)-2-methylpyrrolidine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
piperidine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(2R)-2-(hydroxymethyl)pyrrolidine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(3R)-3-hydroxypyrrolidine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
morpholine-4-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
4-methylpiperazine-1carboxylate; 3-thiazolidinecarboxylic acid
[(2R,3E,5E)-6-[(2R,3S,4E,6R,7R,10R)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
ester;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrro-
lidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-o-
xacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymeth-
yl)pyrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-
-oxo-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-6-(4-meth-
ylpiperazine-1-carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhept-
a-3,5-dienyl] 1,3-dihydroisoindole-2-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-6-(4-meth-
ylpiperazine-1-carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhept-
a-3,5-dienyl] indole-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-[2-(1-hydroxyethyl)p-
yrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-
-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,2-dimethylpyrrolidine-1-carbonyl-
)oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxa-
cyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2S,5S)-2,5-dimethylpyrrolidine-1--
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-o-
xo-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-6-(4-meth-
ylpiperazine-1-carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhept-
a-3,5-dienyl] 2,3-dihydroindole-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-fluoropyrrolidine-1-carbony-
l]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12--
oxo-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-6-(4-meth-
ylpiperazine-1-carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhept-
a-3,5-dienyl] 2-oxa-5-azaspiro[3.4]octane-5-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-6-[6-[(2R)-1-hydroxypropa-
n-2-yl]pyridin-2-yl]hepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclodod-
ec-4-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E)-6-[2-(dimethylamino)pyrimidin-4-yl]hepta-
-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idazin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
imidin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2R,3R,4E,6S,7R,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6R)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-propan-2-ylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-tert-butylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cyclopentylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(oxan-4-yl)piperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
6-cycloheptyl-2,6-diazaspiro[3.3]heptane-2-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptyl-3-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cyclobutylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
N-methyl-N-(1-methylpiperidin-4-yl)carbamate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
morpholine-4-carboxylate;
[(2R,3R,4E,6S,7R,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6R)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
(1S,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
8-cycloheptyl-3,8-diazabicyclo[3.2.1]octane-3-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methyl-1,4-diazepane-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cyclohexylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
piperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptyl-1,4-diazepane-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-hydroxy-6-methylhept-
a-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(azepan-1-yl)piperidine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(8,8-difluoro-3-azabicyclo[3.2.1]octan-3-yl)piperidine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
9-oxo-9-pyrrolidin-1-ylnona-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-y-
l] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-[methyl(propyl)carbamoyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec--
4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrro-
lidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-o-
xacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymeth-
yl)pyrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-
-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10R)-7-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(py-
rrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-10-(pyrrolidine-1-carb-
onyloxy)-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-[(2S)-2-methylpyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-[(3R)-3-methylpyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-[(3R)-3-methylpyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2R)-2-carbamoylpyrrolidine-1-carb-
onyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
-oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-[(2R)-2-(methoxymeth-
yl)pyrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-
-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2S,5S)-2,5-dimethylpyrrolidine-1--
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-o-
xo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-fluoropyrrolidine-1-carbony-
l]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-fluoropyrrolidine-1-carbony-
l]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,2-dimethylpyrrolidine-1-carbonyl-
)oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxa-
cyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10R)-2-[(2E,4E)-6,6-dimethyl-7-(pyrrolidine-1-carbonylox-
y)hepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec--
4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-2-[(2E,4E)-6,6-dimethyl-7-(pyrrolidine-1-carbonylox-
y)hepta-2,4-dien-2-yl]-7-hydroxy-3,7-dimethyl-12-oxo-10-(pyrrolidine-1-car-
bonyloxy)-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
(2R)-1-[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-cycloheptylpiperazine-1-c-
arbonyl)oxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-2-yl]--
2-methylhepta-3,5-dienoxy]carbonylpyrrolidine-2-carboxylic acid;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(3-oxopyrrolidine-1-carbonyl)oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclodo-
dec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-cycloheptylpiperazine-1-carbonyl-
)oxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methy-
lhepta-3,5-dienyl] 2-oxa-7-azaspiro[3.4]octane-7-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-5-[1--
(pyrrolidine-1-carbonyloxymethyl)cyclopropyl]penta-2,4-dien-2-yl]-1-oxacyc-
lododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3S,4R)-3,4-dihydroxypyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12--
oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
(3S)-1-[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-cycloheptylpiperazine-1-c-
arbonyl)oxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-2-yl]--
2-methylhepta-3,5-dienoxy]carbonylpyrrolidine-3-carboxylic acid;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3S)-3-(dimethylamino)pyrrolidine--
1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-
-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,5-dihydropyrrole-1-carbonyloxy)--
6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclo-
dodec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12--
oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-[(3S)-3-[(2-methylpropan-2-yl)oxycarbonylamino]pyrrolidine-1-carbonyl]ox-
yhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-cycloheptylpiperazine-1-carbonyl-
)oxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methy-
lhepta-3,5-dienyl] 3-azabicyclo[3.1.0]hexane-3-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-2-ylhexa-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-(2--
pyrrolidin-1-ylpyrimidin-4-yl)hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6--
yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
azin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E)-6-[2-(dimethylamino)pyrimidin-4-yl]hepta-
-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-(3-methylp-
yridin-2-yl)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-(4-methylp-
yridin-2-yl)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
imidin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idazin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
imidin-4-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyrimidin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyrimidin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-(4-methylp-
yrimidin-2-yl)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-(6--
pyrrolidin-1-ylpyridin-2-yl)hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl-
] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(p-
yrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en--
6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidi-
ne-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacy-
clododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(p-
yrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en--
6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidi-
ne-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacy-
clododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)p-
yrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-
-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-10-hydroxy-3,7-dimethyl-12-oxo--
1-oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-10-hydroxy-3,7-dimethyl-12-oxo--
1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyri-
din-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyri-
din-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyri-
din-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyri-
din-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
N,N-dimethylcarbamate;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-3-[(2-
R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-d-
imethyl-12-oxo-1-oxacyclododec-4-en-6-yl] acetate;
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-carbonylo-
xy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-carbonylo-
xy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6R)-6-(dimethylcarbamoyloxy)-3-methyl-12-oxo-1-o-
xacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
pyrrolidine-1-carboxylate;
[(2R,3E,5E)-6-[(2S,3S,4E,6R)-6-(dimethylcarbamoyloxy)-3-methyl-12-oxo-1-o-
xacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(3R)-3-hydroxypyrrolidine-1-carboxylate;
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-carbonyl]oxy--
6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-[(2S)-2-methylpyrrolidin-
e-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-1-carbo-
nyl]oxy-6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en--
6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-carbonyl]oxy--
6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-1-carbo-
nyl]oxy-6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en--
6-yl] 4-(2,2,2-trifluoroethyl)piperazine-1-carboxylate;
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-
-2-yl]-1-oxacyclododec-4-en-6-yl] acetate;
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-
-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-
-2-yl]-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-
-2-yl]-1-oxacyclododec-4-en-6-yl] N,N-dimethylcarbamate;
[(2S,3S,4E,6R)-2-[(2E,4E)-6-[2-(dimethylamino)pyrimidin-4-yl]hepta-2,4-di-
en-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-(2-pyrrolidin-1-ylpyrimidin-4-
-yl)hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-[2-[(3S)-3-triethylsilyloxypy-
rrolidin-1-yl]pyrimidin-4-yl]hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-y-
l] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-2-[(2E,4E)-6-[2-[(3R)-3-hydroxypyrrolidin-1-yl]pyrimidin-4-
-yl]hepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-pyrimidin-2-ylhepta-2,4-dien--
2-yl]-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrol-
idine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S)-7-hydroxy-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrol-
idine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S)-7-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1--
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclodo-
dec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(3S)-3-
-(1-phenyltetrazol-5-yl)oxypyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]--
12-oxo-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonylamino)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-
-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[[(2R)-2-(hydroxymet-
hyl)pyrrolidine-1-carbonyl]amino]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethy-
l-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonylamino)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-
-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-[methyl(pyrrolidine-1-carbonyl)amino]hepta-2,4-dien-2-yl]-12-oxo-1-oxacy-
clododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(4-cyclopropyltriazol-1-yl)-6-methy-
lhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-methoxycarbonyloxy-6-
-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-9-methoxy-6-methyl-9-o-
xonona-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(cyclopentanecarbonylamino)-6-methy-
lhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(cyclopentanecarbonylamino)-6-methy-
lhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-methylpiperazine-1-carboxylate;
4-cycloheptyl-1-piperazinecarboxylic acid
[(2R,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-7-[ox-
o(1-pyrrolidinyl)methoxy]hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
ester;
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypy-
rrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo--
1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxyl ate;
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-carbonyl]oxy--
6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-azacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2R,3E,5E)-2-methyl-6-[(2S,3S,4E,6R)-3-methyl-6-[(4-methylpiperazine-1-c-
arbonyl)amino]-12-oxo-1-oxacyclododec-4-en-2-yl]hepta-3,5-dienyl]
pyrrolidine-1-carboxylate;
[(2S,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(2R,3R)-3-hydroxy-2-methylpentanoate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-hydroxy-6-methylhept-
a-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-4-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-7-methyl-6-p-
yridin-2-ylocta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-[(2R,3R)-3-[(2R,3R)-3-acetyloxypent-
an-2-yl]oxiran-2-yl]-6-hydroxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-
-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E)-6-hydroxy-6-methyl-8-phen-
yl
octa-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E)-6-hydroxy-6-phen-
ylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E)-6-hydroxy-6-thio-
phen-2-ylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-y-
l] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-phe-
nylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-6-(6-methoxypyridin-2-yl)-
hepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-[-
6-(2-methylpropoxy)pyridin-2-yl]hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclodode-
c-4-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-methyl-8-p-
yridin-2-ylocta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-methyl-7-p-
yridin-2-ylhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E,6R)-6-hydroxy-6-methyl-8-p-
henylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,-
4E)-6-pyridin-2-ylhexa-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-3-ylhexa-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
acetate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-4-ylhexa-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate;
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E)-6-hydroxy-8-(4-hydroxyphe-
nyl)-6-methylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en--
6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-methyl-8-p-
henylocta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-8-[2-(methoxymethyl)pheny-
l]-6-methylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-8-[4-(methoxymethyl)pheny-
l]-6-methylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-8-[3-(methoxymethyl)pheny-
l]-6-methylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate;
[(2S,3S,4E,6S,7S)-7-hydroxy-2-[(2E,4E,6S)-6-hydroxy-6-methyl-7-[(2R,3R)-3-
-[(2S)-3-oxopentan-2-yl]oxiran-2-yl]hepta-2,4-dien-2-yl]-3,7-dimethyl-10,1-
2-dioxo-1-oxacyclododec-4-en-6-yl] acetate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6E,8S)-
-8-pyridin-2-ylnona-2,4,6-trien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methyl-4-oxidopiperazin-4-ium-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(4-fluoropiperidin-1-yl)piperidine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(4,4-difluoropiperidin-1-yl)piperidine-1-carboxylate;
(4S,7S,8S,9E,11S,12S)-4,7,8-trihydroxy-7,11-dimethyl-12-[(2E,4E,6S)-6-pyr-
idin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-9-en-2-one;
[(2S,3S,4E,6S,7S,10S)-7-acetyloxy-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(-
2R,3R)-3-[(2R,3R)-3-
hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-
-12-oxo-1-oxacyclododec-4-en-6-yl] piperazine-1-carboxylate;
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
piperazine-1-carboxylate;
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3-
S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dime-
thyl-12-oxooxacyclododec-4-en-6-yl
piperazine-1-carboxylate;[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-10-hydroxy-2-[-
(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-3-[(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-y-
l]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-7-
-yl] piperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-7-acetyloxy-10-hydroxy-2-[(2E,4E,6R)-7-[(2R,3R)-3-[-
(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-
-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
piperazine-1-carboxylate;
[(2S,3S,4E,6S,7R,10R)-7-ethoxy-10-hydroxy-2-[(2E,4E,6R)-6-hydroxy-7-[(2R,-
3R)-3-[(2S,3S)-3-
hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-
-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-10-hydroxy-2-[(2E,4E,6R)-7-[(2R,3R)-3-[-
(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-
-dimethyl-12-oxo-1-oxacyclododec-4-en-7-yl]
piperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(2R,3R)-3-[(2R,3R)-3-hy-
droxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-methoxy-3,7-d-
imethyl-12-oxo-1-oxacyclododec-4-en-6-yl] piperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(2R,3R)-3-[(2R,3R)-3-hy-
droxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-methoxy-3,7-d-
imethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate;
[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-3-[(2-
R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-met-
hoxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
N-methyl-N[2-(methylamino)ethyl]carbamate;
[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-3-[(2-
R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-met-
hoxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
N-methyl-N[2-(dimethylamino)ethyl]carbamate;
3-[4-[[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-
-3-[(2R,3R)-3-
hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-methoxy-3,-
7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]oxycarbonyl]piperazin-2-yl]pro-
panoic acid;
4-[4-[[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-
-3-[(2R,3R)-3-
hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-methoxy-3,-
7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]oxycarbonyl]piperazin-1-yl]but-
anoic acid;
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate;
(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-yl2,5-diazabicyclo[2.2.1]heptane-
-2-carboxylate;
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-propylpiperazine-1-carboxyla-
te;
(2R,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((2S,6R,E)-6-hydroxy-7-((2R-
,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhept-4-en-2-yl)--
3,7-di
methyl-12-oxooxacyclododec-4-en-7-yl4-(2-hydroxyethyl)piperazine-1--
carboxylate;
(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-yl4-methylpiperazine-1-carboxyla-
te;
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R-
,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-
-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl4-(2-aminoethyl)piperazine--
1-carboxylate;
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
4-(2-ethoxy-2-oxoethyl)piperazine-1-carboxylate;
(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-(R,2E,4E)-6-hydroxy-7-(2R,3R)-3-((2R-
,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-di-
methyl-12-oxooxacyclododec-4-en-6-yl
4-methylpiperazine-1-carboxylate;
(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-(R,2E,4E)-6-hydroxy-7-(2R,3R)-3-((2R-
,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-di-
methyl-12-oxooxacyclododec-4-en-6-yl piperazine-1-carboxylate; and
pharmaceutically acceptable salts thereof.
9. The compound of any one of claims 1-8, wherein said compound is
stereomerically pure.
10. A pharmaceutical composition comprising a compound and/or
pharmaceutically acceptable salt according to any one of claims
1-9.
11. The pharmaceutical composition of claim 10, wherein said
composition is formulated for intravenous, oral, subcutaneous, or
intramuscular administration.
12. A method of treating cancer in a subject in need thereof,
comprising administering to said subject a therapeutically
effective amount of a compound and/or pharmaceutically acceptable
salt according to any one of claims 1-9, or a pharmaceutical
composition according to claim 10 or claim 11.
13. The method of claim 12, wherein said cancer is chosen from
myelodysplastic syndrome, chronic lymphocytic leukemia, chronic
myelomonocytic leukemia, acute myeloid leukemia, colon cancer,
pancreatic cancer, endometrial cancer, ovarian cancer, breast
cancer, uveal melanoma, gastric cancer, cholangiocarcinoma, and
lung cancer.
14. The method of claim 13, wherein said cancer is chosen from
myelodysplastic syndrome, chronic lymphocytic leukemia, chronic
myelomonocytic leukemia, and acute myeloid leukemia.
15. The method of claim 13, wherein said cancer is myelodysplastic
syndrome.
16. The method of claim 13, wherein said cancer is chronic
lymphocytic leukemia.
17. The method of claim 13, wherein said cancer is chronic
myelomonocytic leukemia
18. The method of claim 13, wherein said cancer is acute myeloid
leukemia.
19. The method of claim 13, wherein said cancer is colon
cancer.
20. The method of claim 13, wherein said cancer is pancreatic
cancer.
21. The method of claim 13, wherein said cancer is endometrial
cancer.
22. The method of claim 13, wherein said cancer is ovarian
cancer.
23. The method of claim 13, wherein said cancer is breast
cancer.
24. The method of claim 13, wherein said cancer is uveal
melanoma.
25. The method of claim 13, wherein said cancer is gastric
cancer.
26. The method of claim 13, wherein said cancer is
cholangiocarcinoma.
27. The method of claim 13, wherein said cancer is lung cancer.
28. The method of any one of claims 12-27, wherein said cancer is
positive for one or more mutations in a spliceosome gene or
protein.
29. The method of claim 28, wherein said spliceosome gene or
protein is chosen from splicing factor 3B subunit 1 (SF3B1), U2
small nuclear RNA auxiliary factor 1 (U2AF1), serine/arginine-rich
splicing factor 2 (SRSF2), zinc finger (CCCH type) RNA-binding
motif and serine/arginine rich 2 (ZRSR2),
pre-mRNA-processing-splicing factor 8 (PRPF8), U2 small nuclear RNA
auxiliary factor 2 (U2AF2), splicing factor 1 (SF1), splicing
factor 3a subunit 1 (SF3A1), PRP40 pre-mRNA processing factor 40
homolog B (PRPF40B), RNA binding motif protein 10 (RBM10), poly(rC)
binding protein 1 (PCBP1), crooked neck pre-mRNA splicing factor 1
(CRNKL1), DEAH (Asp-Glu-Ala-His) box helicase 9 (DHX9),
peptidyl-prolyl cis-trans isomerase-like 2 (PPIL2), RNA binding
motif protein 22 (RBM22), small nuclear ribonucleoprotein Sm D3
(SNRPD3), probable ATP-dependent RNA helicase DDX5 (DDX5),
pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 (DHX15),
and polyadenylate-binding protein 1 (PABPC1).
30. The method of claim 29, wherein said spliceosome gene or
protein is Splicing Factor 3B subunit 1 (SF3B1).
31. The use of the compound and/or pharmaceutically acceptable salt
according to any one of claims 1-9, or the pharmaceutical
composition according to claim 10 or claim 11, in the preparation
of a medicament for the treatment of cancer.
32. The use of claim 31, wherein said cancer is chosen from
myelodysplastic syndrome, chronic lymphocytic leukemia, chronic
myelomonocytic leukemia, acute myeloid leukemia, colon cancer,
pancreatic cancer, endometrial cancer, ovarian cancer, breast
cancer, uveal melanoma, gastric cancer, cholangiocarcinoma, and
lung cancer.
33. The use of claim 32, wherein said cancer is chosen from
myelodysplastic syndrome, chronic lymphocytic leukemia, chronic
myelomonocytic leukemia, and acute myeloid leukemia.
34. The use of claim 32, wherein said cancer is myelodysplastic
syndrome.
35. The use of claim 32, wherein said cancer is chronic lymphocytic
leukemia.
36. The use of claim 32, wherein said cancer is chronic
myelomonocytic leukemia
37. The use of claim 32, wherein said cancer is acute myeloid
leukemia.
38. The use of claim 32, wherein said cancer is colon cancer.
39. The use of claim 32, wherein said cancer is pancreatic
cancer.
40. The use of claim 32, wherein said cancer is endometrial
cancer.
41. The use of claim 32, wherein said cancer is ovarian cancer.
42. The use of claim 32, wherein said cancer is breast cancer.
43. The use of claim 32, wherein said cancer is uveal melanoma.
44. The use of claim 32, wherein said cancer is gastric cancer.
45. The use of claim 32, wherein said cancer is
cholangiocarcinoma.
46. The use of claim 32, wherein said cancer is lung cancer.
47. The use of any one of claims 31-46, wherein said cancer is
positive for one or more mutations in a spliceosome gene or
protein.
48. The use of claim 47, wherein said spliceosome gene or protein
is chosen from splicing factor 3B subunit 1 (SF3B1), U2 small
nuclear RNA auxiliary factor 1 (U2AF1), serine/arginine-rich
splicing factor 2 (SRSF2), zinc finger (CCCH type) RNA-binding
motif and serine/arginine rich 2 (ZRSR2),
pre-mRNA-processing-splicing factor 8 (PRPF8), U2 small nuclear RNA
auxiliary factor 2 (U2AF2), splicing factor 1 (SF1), splicing
factor 3a subunit 1 (SF3A1), PRP40 pre-mRNA processing factor 40
homolog B (PRPF40B), RNA binding motif protein 10 (RBM10), poly(rC)
binding protein 1 (PCBP1), crooked neck pre-mRNA splicing factor 1
(CRNKL1), DEAH (Asp-Glu-Ala-His) box helicase 9 (DHX9),
peptidyl-prolyl cis-trans isomerase-like 2 (PPIL2), RNA binding
motif protein 22 (RBM22), small nuclear ribonucleoprotein Sm D3
(SNRPD3), probable ATP-dependent RNA helicase DDX5 (DDX5),
pre-mRNA-splicing factor ATP-dependent RNA helicase DHX15 (DHX15),
and polyadenylate-binding protein 1 (PABPC1).
49. The use of claim 48, wherein said spliceosome gene or protein
is Splicing Factor 3B subunit 1 (SF3B1).
50. A method of treating cancer in a subject in need thereof,
comprising administering to said subject a therapeutically
effective amount of a compound and/or pharmaceutically acceptable
salt according to any one of claims 1-9, or a pharmaceutical
composition according to claim 10 or claim 11; and at least one
additional therapy.
51. The method of claim 50, wherein the at least one additional
therapy comprises at least one, at least two, at least three, at
least four, or at least five additional therapies.
52. The method of claim 50, wherein the administered amount of the
compound and/or pharmaceutically acceptable salt according to any
one of claims 1-9, or a pharmaceutical composition according to
claim 10 or claim 11, and/or the at least one additional therapy is
reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or
90%, relative to a standard dosage of the compound and/or
pharmaceutically acceptable salt according to any one of claims
1-9, or a pharmaceutical composition according to claim 10 or claim
11, and/or the at least one additional therapy.
53. The method of any one of claims 50 to 52, wherein the compound
and/or pharmaceutically acceptable salt according to any one of
claims 1-9, or a pharmaceutical composition according to claim 10
or claim 11, and/or the at least one additional therapy is
administered at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
75%, or 90% less frequently, relative to a standard dosing regimen
of the compound and/or pharmaceutically acceptable salt according
to any one of claims 1-9, or a pharmaceutical composition according
to claim 10 or claim 11, and/or the at least one additional
therapy.
54. The method of any one of claims 50 to 53, wherein the
administered amount and/or dosage of the compound and/or
pharmaceutically acceptable salt according to any one of claims
1-9, or the pharmaceutical composition according to claim 10 or
claim 11, and/or the at least one additional therapy results in
lower systemic toxicity and/or improved tolerance.
55. The method of claim 50, wherein administration of the compound
and/or pharmaceutically acceptable salt according to any one of
claims 1-9, or the pharmaceutical composition according to claim 10
or claim 11, is initiated before administration of the at least one
additional therapy.
56. The method of claim 50, wherein administration of the compound
and/or pharmaceutically acceptable salt according to any one of
claims 1-9, or the pharmaceutical composition according to claim 10
or claim 11, is initiated after administration of the at least one
additional therapy.
57. The method of claim 50, wherein administration of the compound
and/or pharmaceutically acceptable salt according to any one of
claims 1-9, or the pharmaceutical composition according to claim 10
or claim 11, is initiated concurrently with administration of the
at least one additional therapy.
58. The method of any one of claims 50 to 57, wherein
administration of the compound and/or pharmaceutically acceptable
salt according to any one of claims 1-9, or the pharmaceutical
composition according to claim 10 or claim 11, is repeated at least
once after initial administration.
59. The method of claim 58, wherein the amount of the compound
and/or pharmaceutically acceptable salt according to any one of
claims 1-9, or the pharmaceutical composition according to claim 10
or claim 11, used for repeated administration is reduced relative
to the amount used for initial administration.
60. The method of claim 58, wherein the amount of the compound
and/or pharmaceutically acceptable salt according to any one of
claims 1-9, or the pharmaceutical composition according to claim 10
or claim 11, used for repeated administration is reduced relative
to a standard dosage of the compound and/or pharmaceutically
acceptable salt according to any one of claims 1-9, or the
pharmaceutical composition according to claim 10 or claim 11.
61. The method of claim 58, wherein the amount of the compound
and/or pharmaceutically acceptable salt according to any one of
claims 1-9, or the pharmaceutical composition according to claim 10
or claim 11, used for repeated administration is reduced by 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, relative to a
standard dosage of the compound and/or pharmaceutically acceptable
salt according to any one of claims 1-9, or the pharmaceutical
composition according to claim 10 or claim 11.
62. The method of any one of claims 50 to 61, wherein
administration of the at least one additional therapy is repeated
at least once after initial administration.
63. The method of claim 62, wherein the amount of the at least one
additional therapy used for repeated administration is reduced
relative to the amount used for initial administration.
64. The method of claim 62, wherein the amount of the at least one
additional therapy used for repeated administration is reduced
relative to a standard dosage of the at least one additional
therapy.
65. The method of claim 62, wherein the amount of the at least one
additional therapy used for repeated administration is reduced by
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, relative
to a standard dosage of the at least one additional therapy.
66. The method of any one of claims 50 to 65, wherein repeated
administration of the compound and/or pharmaceutically acceptable
salt according to any one of claims 1-9, or the pharmaceutical
composition according to claim 10 or claim 11, is concurrent with
repeated administration of the at least one additional therapy.
67. The method of any one of claims 50 to 65, wherein repeated
administration of the compound and/or pharmaceutically acceptable
salt according to any one of claims 1-9, or the pharmaceutical
composition according to claim 10 or claim 11, is sequential or
staggered with repeated administration of the at least one
additional therapy.
68. The method of any one of claims 50 to 67, wherein the at least
one additional therapy comprises administering a checkpoint
inhibitor.
69. The method of claim 68, wherein the subject is intolerant,
non-responsive, or poorly responsive to the checkpoint inhibitor
when administered alone.
70. The method of claim 68, wherein the checkpoint inhibitor
targets CTLA4, PD1, PDL1, OX40, CD40, GITR, LAG3, TIM3, and/or
KIR.
71. The method of claim 68, wherein the checkpoint inhibitor
targets CTLA4, OX40, CD40, and/or GITR.
72. The method of claim 70 or claim 71, wherein the checkpoint
inhibitor comprises a cytotoxic T-lymphocyte-associated antigen 4
pathway (CTLA4) inhibitor.
73. The method of claim 72, wherein the CTLA4 inhibitor is an
anti-CTLA4 antibody.
74. The method of claim 73, wherein the anti-CTLA4 antibody is
ipilimumab.
75. The method of claim 70 or claim 71, wherein the checkpoint
inhibitor comprises a programmed death-1 pathway (PD1)
inhibitor.
76. The method of claim 75, wherein the PD1 inhibitor is an
anti-PD1 antibody.
77. The method of claim 76, wherein the anti-PD1 antibody is
nivolumab.
78. The method of claim 75, wherein the PD1 inhibitor is an
anti-PDL1 antibody.
79. The method of claim 78, wherein the anti-PDL1 antibody is
atezolizumab.
80. The method of claim 70 or claim 71, wherein the checkpoint
inhibitor comprises a CTLA4 inhibitor and a PD1 inhibitor.
81. The method of claim 80, wherein the CTLA4 inhibitor is an
anti-CTLA4 antibody.
82. The method of claim 81, wherein the anti-CTLA4 antibody is
ipilimumab.
83. The method of claim 80 or claim 81, wherein the PD1 inhibitor
is an anti-PD1 antibody.
84. The method of claim 83, wherein the anti-PD1 antibody is
nivolumab.
85. The method of claim 80 or claim 81, wherein the PD1 inhibitor
is an anti-PDL1 antibody.
86. The method of claim 85, wherein the anti-PDL1 antibody is
atezolizumab.
87. The method of any one of claims 50 to 67, wherein the at least
one additional therapy comprises administering a cytokine or
cytokine analog.
88. The method of claim 87, wherein the subject is intolerant,
non-responsive, or poorly responsive to the cytokine or cytokine
analog when administered alone.
89. The method of claim 87, wherein the cytokine or cytokine analog
comprises a T-cell enhancer.
90. The method of claim 87, wherein the cytokine or cytokine analog
comprises IL-2, IL-10, IL-12, IL-15, IFN.gamma., and/or
TNF.alpha..
91. The method of any one of claims 50 to 67, wherein the at least
one additional therapy comprises administering engineered
tumor-targeting T-cells.
92. The method of any one of claims 50 to 91, wherein the subject
has a non-synonymous mutational burden of about 150 mutations or
less.
93. The method of any one of claims 50 to 92, wherein the subject
has a non-synonymous mutational burden of about 100 mutations or
less.
94. The method of any one of claims 50 to 93, wherein the subject
has a non-synonymous mutational burden of about 50 mutations or
less.
95. The method of any one of claims 50 to 94, wherein the cancer is
a hematological malignancy or a solid tumor.
96. The method of claim 95, wherein the hematological malignancy is
chosen from a B-cell malignancy, a leukemia, a lymphoma, and a
myeloma.
97. The method of claim 95 or claim 96, wherein the hematological
malignancy is chosen from acute myeloid leukemia and multiple
myeloma.
98. The method of claim 95, wherein the solid tumor is chosen from
breast cancer, gastric cancer, prostate cancer, ovarian cancer,
lung cancer, uterine cancer, salivary duct carcinoma, melanoma,
colon cancer, and esophageal cancer.
99. The method of any one of claims 50 to 94, wherein the cancer is
chosen from myelodysplastic syndrome, chronic lymphocytic leukemia,
acute lymphoblastic leukemia, chronic myelomonocytic leukemia,
acute myeloid leukemia, colon cancer, pancreatic cancer,
endometrial cancer, ovarian cancer, breast cancer, uveal melanoma,
gastric cancer, cholangiocarcinoma, and lung cancer.
100. A method of inducing at least one neoantigen, comprising
contacting a neoplastic cell with a therapeutically effective
amount of a compound and/or pharmaceutically acceptable salt
according to any one of claims 1-9, or a pharmaceutical composition
according to claim 10 or claim 11, thereby inducing production of
at least one neoantigen.
101. The method of claim 100, wherein the neoplastic cell is
present in an in vitro cell culture.
102. The method of claim 100 or claim 101, wherein the neoplastic
cell is obtained from a subject.
103. The method of claim 100, wherein the neoplastic cell is
present in a subject.
104. The method of any one of claims 100 to 103, wherein the
neoplastic cell is derived from a hematological malignancy or a
solid tumor.
105. The method of claim 104, wherein the hematological malignancy
is selected from a B-cell malignancy, a leukemia, a lymphoma, and a
myeloma.
106. The method of claim 104 or claim 105, wherein the
hematological malignancy is selected from acute myeloid leukemia
and multiple myeloma.
107. The method of claim 104, wherein the solid tumor is selected
from breast cancer, gastric cancer, prostate cancer, ovarian
cancer, lung cancer, uterine cancer, salivary duct carcinoma,
melanoma, colon cancer, and esophageal cancer.
108. A method of inducing at least one neoantigen and/or a T-cell
response in a subject having or suspected of having a neoplastic
disorder, comprising administering to the subject a therapeutically
effective amount of a compound and/or pharmaceutically acceptable
salt according to any one of claims 1-9, or a pharmaceutical
composition according to claim 10 or claim 11.
109. A method of treating a subject having or suspected of having a
neoplastic disorder, comprising administering to the subject a
therapeutically effective amount of a compound and/or
pharmaceutically acceptable salt according to any one of claims
1-9, or a pharmaceutical composition according to claim 10 or claim
11, wherein administration of the compound and/or pharmaceutically
acceptable salt, or pharmaceutical composition, induces at least
one neoantigen and/or a T-cell response.
110. The method of claim 109, wherein the amount of the compound
and/or pharmaceutically acceptable salt, or pharmaceutical
composition, administered is reduced due to induction of at least
one neoantigen and/or a T-cell response, relative to a standard
dosage of the compound and/or pharmaceutically acceptable salt, or
pharmaceutical composition.
111. The method of claim 110, wherein the administered amount of
the compound and/or pharmaceutically acceptable salt, or
pharmaceutical composition, is reduced by 10%, 15%, 20%, 25%, 30%,
35%, 40%, 45%, 50%, 75%, or 90%, relative to a standard dosage of
the compound and/or pharmaceutically acceptable salt, or
pharmaceutical composition.
112. The method of any one of claims 109 to 111, wherein the
compound and/or pharmaceutically acceptable salt, or pharmaceutical
composition, is administered at least 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 75%, or 90% less frequently, relative to a standard
dosing regimen of the compound and/or pharmaceutically acceptable
salt, or pharmaceutical composition.
113. The method of any one of claims 109 to 111, wherein the
administered amount and/or dosage of the compound and/or
pharmaceutically acceptable salt, or pharmaceutical composition,
results in lower systemic toxicity and/or improved tolerance.
114. The method of any one of claims 108 to 113, further comprising
administering at least one additional therapy.
Description
[0001] The present application claims the benefit of priority to
U.S. Provisional Application No. 62/655,021 filed Apr. 9, 2018;
U.S. Provisional Application No. 62/679,653 filed Jun. 1, 2018;
U.S. Provisional Application No. 62/814,838 filed Mar. 6, 2019; and
U.S. Provisional Application No. 62/814,843 filed Mar. 6, 2019, all
of which are incorporated herein by reference.
[0002] Disclosed herein are novel organic compounds and
pharmaceutical compositions containing such compounds. These
compounds are useful in the treatment of cancer, particularly
cancers in which agents that target the spliceosome and mutations
therein are known to be useful. These compounds are also useful in
the treatment of cancer when administered in combination with at
least one additional therapy.
[0003] In eukaryote organisms, newly synthesized messenger RNAs
typically have multiple introns, which are excised to provide the
mature mRNA. The spliceosome is a multisubunit complex that
accomplishes this task. The spliceosome consists of five small
nuclear RNAs (snRNAs; U1-6) in combination with a variety of
proteins.
[0004] Mutations in the splicing factor 3B subunit 1 (SF3B1) of the
spliceosome exist in a number of cancers and comprise a target for
anticancer agents. Compounds isolated from the bacteria
Streptomyces platensis (Sakai, Takashi; Sameshima, Tomohiro;
Matsufuji, Motoko; Kawamura, Naoto; Dobashi, Kazuyuki; Mizui,
Yoshiharu. Pladienolides, New Substances from Culture of
Streptomyces platensis Mer-11107. I. Taxonomy, Fermentation,
Isolation and Screening. The Journal of Antibiotics. 2004, Vol. 57,
No.3.), termed pladienolides and discovered while screening for
inhibitors of the vascular endothelial growth factor (VEGF)
promoter, inhibit expression of a reporter gene controlled by human
VEGF promoter, which inhibition is known to be a useful mechanism
of action for anticancer agents.
[0005] These compounds also inhibit proliferation of U251 human
glioma cells in vitro. The most potent of these compounds,
Pladienolide B, inhibits VEGF-promoted gene expression with an ICso
of 1.8 nM, and inhibits glioma cell proliferation with an ICso of
3.5 nM. The structure of pladienolide B is known, (Sakai, Takashi;
Sameshima, Tomohiro; Matsufuji, Motoko; Kawamura, Naoto; Dobashi,
Kazuyuki; Mizui, Yoshiharu. Pladienolides, New Substances from
Culture of Streptomyces platensis Mer-11107. II. Physico-chemical
Properties and Structure Elucidation. The Journal of Antibiotics.
Vol. 57, No.3. (2004)) and pladienolide B is known to target the
SF3b spliceosome to inhibit splicing and alter the pattern of gene
expression (Kotake et al., "Splicing factor SF3b as a target of the
antitumor natural product pladienolide", Nature Chemical Biology
2007, 3, 570-575).
[0006] Certain pladienolide B analogs are, likewise, known: WO
2002/060890; WO 2004/011459; WO 2004/011661; WO 2004/050890; WO
2005/052152; WO 2006/009276; WO 2008/126918; and WO 2015/175594.
For example, a pladienolide compound,
(8E,12E,14E)-7-((4-Cycoheptylpiperazin-1-yl)carbonyl)oxy-3,6,16,21-tetrah-
ydroxy-6,10,12,16,20-pentamethyl-18,19-epoxytricosa-8,12,14-trien-11-olide-
, also known as E7107, is a semisynthetic derivative of the natural
product pladienolide D, and the results of its Phase I study have
been reported. As another example, the pladienolide pyridine
compound
(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-4R,2E,4E)-6-(pyr-
idin-2-yl)hepta-2,4-dien-2-yl)oxacyclododec-4-en-6-yl
4-methylpiperazine-1-carboxylate (also named
"(2S,3S,4E,6S,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-((2E,4E,6R)-6--
(pyridin-2-yl)hepta-2,4-dien-2-yl)oxacyclododec-4-en-6-yl
4-methylpiperazine-1-carboxylate"), also known as H3B-8800, has
received orphan drug designation for the treatment of certain
hematological cancers.
[0007] However, additional agents useful in the treatment of
cancer, particularly cancers in which agents that target the
spliceosome and mutations therein are known to be useful, are
needed. Immune checkpoint blockade (ICB) has recently proven to be
a paradigm shift for the treatment of several different cancer
types. However, not all patients demonstrate robust/durable
responses to ICB. See, e.g., Zappasodi, R. et al. Emerging Concepts
for Immune Checkpoint Blockade-Based Combination Therapies. Cancer
Cell 33, 581-598, doi:10.1016/j.ccell.2018.03.005 (2018); and
Wolchok, J. D. et al. Overall Survival with Combined Nivolumab and
Ipilimumab in Advanced Melanoma. N Engl J Med 377, 1345-1356,
doi:10.1056/NEJMoal709684 (2017). Therefore, there also exists a
need to discover complementary therapeutic agents to administer in
combination with ICB or any other therapy to improve and/or
maximize patient response.
[0008] Disclosed herein are compounds of Formula I:
##STR00001##
and pharmaceutically acceptable salts thereof, [0009] wherein:
[0010] n is chosen from 0, 1, 2 and 3;
[0011] R.sup.1 is chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, --NR.sup.9R.sup.10 groups,
##STR00002##
groups,
##STR00003##
groups,
##STR00004##
groups,
##STR00005##
groups,
##STR00006##
groups,
##STR00007##
groups
##STR00008##
groups, and
##STR00009##
groups;
[0012] R.sup.9 is chosen from hydrogen, --NR.sup.11R.sup.12 groups,
C.sub.1-C.sub.6 alkyl groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H
groups, C.sub.3-C.sub.8 cycloalkyl groups, and C.sub.3-C.sub.8
heterocyclyl groups, wherein the C.sub.3-C.sub.8 cycloalkyl groups
and C.sub.3-C.sub.8 heterocyclyl groups may be unsubstituted or
substituted from 1-3 times with a group independently chosen from
C.sub.1-C.sub.6 alkyl groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H
groups, hydroxy, halogen groups, and C.sub.1-C.sub.6 alkoxy
groups;
[0013] R.sup.10 is chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups;
[0014] one of either R.sup.2 or R.sup.3 is chosen from hydrogen and
C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, --OR.sup.10, --OC(O)R.sup.10, --OC(O)R.sup.1, and
C.sub.1-C.sub.6 alkyl groups;
[0015] R.sup.4 is hydrogen or hydroxy;
[0016] R.sup.5 and R.sup.6 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups;
[0017] R7 and R.sup.8 are each independently chosen from hydrogen,
hydroxy, C.sub.1-C.sub.6 alkoxy groups, and C.sub.1-C.sub.6 alkyl
groups; and
[0018] Y is chosen from phenyl, thiophenyl, triazolyl, pyridinyl,
pyrimidinyl, pyridazinyl, and pyrazinyl, wherein Y may be
unsubstituted or substituted from 1-3 times with a group
independently chosen from hydroxyl, oxo groups, C.sub.1-C.sub.6
alkyl groups, C.sub.3-C.sub.5 cycloalkyl groups, hydroxy
C.sub.1-C.sub.6 alkyl groups, C.sub.1-C.sub.6 alkoxy groups,
methoxy C.sub.1-C.sub.6 alkyl groups, --NR.sup.11R.sup.12
groups,
##STR00010##
wherein R.sup.11 and R.sup.12 are each independently chosen from
hydrogen and C.sub.1-C.sub.6 alkyl groups.
[0019] Also disclosed herein are compounds of Formula II:
##STR00011##
and pharmaceutically acceptable salts thereof, [0020] wherein:
[0021] X is chosen from O, NR' groups, and CH.sub.2, wherein R' is
chosen from hydrogen and C.sub.1-C.sub.6 alkyl groups;
[0022] R.sup.1 is chosen from methyl, NR.sup.11R.sup.12 groups,
##STR00012##
groups, and
##STR00013##
groups,
[0023] R.sup.10 is chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, and halo-C.sub.1-C.sub.6 alkyl
groups, wherein the C.sub.3-C.sub.8 cycloalkyl groups may be
unsubstituted or substituted from 1-3 times with a group
independently chosen from C.sub.1-C.sub.6 alkyl groups, hydroxy,
halogen groups, and C.sub.1-C.sub.6 alkoxy groups;
[0024] R.sup.11 and R.sup.12 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups;
[0025] one of either R.sup.2 or R.sup.3 is hydrogen or
C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, hydroxy and C.sub.1-C.sub.6 alkyl groups;
[0026] one of either R.sup.4 or R.sup.5 is hydrogen, and the other
is chosen from hydrogen, hydroxy, and
##STR00014##
[0027] R.sup.6 and R.sup.7 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups; R.sup.8 and R.sup.9 are each
independently chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups; or R.sup.8 and R.sup.9 are taken together to form a
cyclopropyl ring; and
[0028] Y is chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, methoxy, and --NR.sup.13R.sup.14
groups, wherein R.sup.13 and R.sup.14 are each independently chosen
from hydrogen, C.sub.1-C.sub.6 alkyl groups, and methoxy
C.sub.1-C.sub.6 alkyl groups; or R.sup.13 and R.sup.14 may be taken
together with the N to form a group chosen from
##STR00015##
a morpholine, a piperidine, a thiazolidine, an indole, an indoline,
and an isoindoline ring;
[0029] wherein Y may be unsubstituted or substituted from 1-3 times
with a group independently chosen from C.sub.1-C.sub.6 alkyl
groups, hydroxy, hydroxy C.sub.1-C.sub.6 alkyl groups, methoxy,
methoxy C.sub.1-C.sub.6 alkyl groups, halo, halo C.sub.1-C.sub.6
alkyl groups, --C(O)NH.sub.2, --NHCOO--C.sub.1-C.sub.6 alkyl
groups, --COOH,
##STR00016##
and --NR.sup.15R.sup.16 groups, wherein R.sup.15 and R.sup.16 are
each independently chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups.
[0030] Also disclosed herein are compounds of Formula III:
##STR00017##
and pharmaceutically acceptable salts thereof, [0031] wherein:
[0032] n is chosen from 0, 1, 2 and 3;
[0033] m is chosen from 1, 2, and 3;
[0034] R.sup.1 is chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, --NR.sup.11R.sup.12 groups,
##STR00018##
groups,
##STR00019##
groups,
##STR00020##
groups,
##STR00021##
groups,
##STR00022##
groups,
##STR00023##
groups,
##STR00024##
groups,
##STR00025##
groups, and
##STR00026##
groups;
[0035] R.sup.11 is chosen from hydrogen, --NR.sup.16R.sup.17
groups, C.sub.1-C.sub.6 alkyl groups, C.sub.3-C.sub.8 cycloalkyl
groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H groups,
--(C.sub.1-C.sub.6 alkyl)--CO.sub.2R.sup.12 groups,
--(C.sub.1-C.sub.6 alkyl)--NR.sup.16R.sup.17 groups, and
C.sub.3-C.sub.8 heterocyclyl groups, wherein the
--NR.sup.11R.sup.12 groups, C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, and C.sub.3-C.sub.8 heterocyclyl
groups may be unsubstituted or substituted from 1-3 times with a
group independently chosen from C.sub.1-C.sub.6 alkyl groups,
--(C.sub.1-C.sub.6 alkyl)--CO.sub.2H groups, hydroxy, halogen
groups, and C.sub.1-C.sub.6 alkoxy groups;
[0036] R.sup.12 is chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups;
[0037] one of either R.sup.2 or R.sup.3 is chosen from hydrogen and
C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, --OR.sup.10, --PC(O)E.sup.10, --OC(O)R.sup.1 and
C.sub.1-C.sub.6 alkyl groups;
[0038] R.sup.4 is chosen from hydrogen and hydroxy;
[0039] R.sup.5 and R.sup.6 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups;
[0040] R.sup.7 and R.sup.8 are each independently chosen from
hydrogen, hydroxy, C.sub.1-C.sub.6 alkoxy groups, and
C.sub.1-C.sub.6 alkyl groups; and
[0041] R.sup.9 and R.sup.10 are each independently chosen from
hydrogen, C.sub.1-C.sub.6 alkyl groups, hydroxy, and
C.sub.1-C.sub.6 alkoxy groups; or, one of R.sup.9 or R.sup.10 is
oxo and the other is absent;
[0042] Z is chosen from C.sub.1-C.sub.6 alkyl groups,
--C(O)--C.sub.1-C.sub.6 alkyl groups, --OR.sup.13, and
--NR.sup.14R.sup.15 groups,
[0043] wherein R.sup.13 is chosen from hydrogen, C.sub.1-C.sub.6
alkyl groups, and --C(O)--C.sub.1-C.sub.6 alkyl groups,
[0044] wherein R.sup.14 and R.sup.15 are each independently chosen
from hydrogen, C.sub.1-C.sub.6 alkyl groups, and methoxy
C.sub.1-C.sub.6 alkyl groups; or R.sup.14 and R.sup.15 may be taken
together with the N to form a group chosen from
##STR00027##
a morpholine, a piperidine, a thiazolidine, an indole, an indoline,
and an isoindoline ring;
[0045] wherein Z may be unsubstituted or substituted from 1-3 times
with a group independently chosen from C.sub.1-C.sub.6 alkyl
groups, C.sub.3-C.sub.5 cycloalkyl groups, hydroxy C.sub.1-C.sub.6
alkyl groups, C.sub.1-C.sub.6 alkoxy groups, methoxy
C.sub.1-C.sub.6 alkyl groups, --NR.sup.16R.sup.17 groups,
##STR00028##
wherein R.sup.16 and R.sup.17 are each independently chosen from
hydrogen and C.sub.1-C.sub.6 alkyl groups.
[0046] Also disclosed herein are pharmaceutical compositions
comprising at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, the pharmaceutical compositions further comprise at
least one pharmaceutically acceptable carriers.
[0047] Also disclosed herein are methods of treating a subject with
cancer comprising administering to the subject a therapeutically
acceptable amount of at least one compound of Formula I, at least
one compound of Formula II, at least one compound of Formula III,
and/or a pharmaceutically acceptable salt of any of the foregoing.
In some embodiments, the cancer may be chosen from myelodysplastic
syndrome, chronic lymphocytic leukemia, chronic myelomonocytic
leukemia, acute myeloid leukemia, colon cancer, pancreatic cancer,
endometrial cancer, ovarian cancer, breast cancer, uveal melanoma,
gastric cancer, cholangiocarcinoma, and/or lung cancer. In some
embodiments, the cancer is chosen from cancers that test positive
for one or more mutations in the Splicing factor 3B subunit 1
(SF3B1) gene or protein. In some embodiments, the cancer is chosen
from cancers that test positive for one or more mutations in a
spliceosome gene or protein, such as those listed in Table 1. In
some embodiments, administration of at least one compound of
Formula I, at least one compound of Formula II, at least one
compound of Formula III, and/or a pharmaceutically acceptable salt
of any of the foregoing, induces at least one neoantigen and/or a
T-cell response.
[0048] Also disclosed herein are uses of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing in a method of therapeutic treatment, e.g.,
treatment for a cancer. In some embodiments, the cancer may be
chosen from myelodysplastic syndrome, chronic lymphocytic leukemia,
chronic myelomonocytic leukemia, acute myeloid leukemia, colon
cancer, pancreatic cancer, endometrial cancer, ovarian cancer,
breast cancer, uveal melanoma, gastric cancer, cholangiocarcinoma,
and/or lung cancer. In some embodimentms, the cancer is chosen from
cancers that test positive for one or more mutations in the
Splicing factor 3B subunit 1 (SF3B1) gene or protein. In some
embodiments, the cancer is chosen from cancers that test positive
for one or more mutations in a spliceosome gene or protein, such as
those listed in Table 1. In some embodiments, administration of at
least one compound of Formula I, at least one compound of Formula
II, at least one compound of Formula III, and/or a pharmaceutically
acceptable salt of any of the foregoing, induces at least one
neoantigen and/or a T-cell response.
[0049] Also disclosed herein are uses of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing, in the preparation of a medicament. In some
embodiments, the medicament is useful for the treatment of cancer.
In some embodiments, the cancer may be chosen from myelodysplastic
syndrome, chronic lymphocytic leukemia, chronic myelomonocytic
leukemia, acute myeloid leukemia, colon cancer, pancreatic cancer,
endometrial cancer, ovarian cancer, breast cancer, uveal melanoma,
gastric cancer, cholangiocarcinoma, and/or lung cancer. In some
embodiments, the cancer is chosen from cancers that test positive
for one or more mutations in the Splicing factor 3B subunit 1
(SF3B1) gene or protein. In some embodiments, the cancer is chosen
from cancers that test positive for one or more mutations in a
spliceosome gene or protein, such as those listed in Table 1. In
some embodiments, administration of at least one compound of
Formula I, at least one compound of Formula II, at least one
compound of Formula III, and/or a pharmaceutically acceptable salt
of any of the foregoing, induces at least one neoantigen and/or a
T-cell response.
[0050] Also disclosed herein are uses of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing to target the spliceosome, e.g., subunit 1 of
the SF3B spliceosome. As used herein, the following definitions
shall apply unless otherwise indicated.
[0051] Also disclosed herein are methods of inducing at least one
neoantigen, comprising contacting a neoplastic cell with a
therapeutically effective amount at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing. In some embodiments, such contact may induce production
of at least one neoantigen.
[0052] Also disclosed herein are methods of inducing at least one
neoantigen and/or a T-cell response in a subject having or
suspected of having a neoplastic disorder, comprising administering
to the subject a therapeutically effective amount of at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing.
[0053] Also disclosed herein are methods of treating a subject
having or suspect of having a neoplastic disorder. In some
embodiments, the method comprises administering to the subject a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, wherein administration may result in inducing at least
one neoantigen and/or a T-cell response. In some embodiments, the
method may also comprise detecting one or more neoantigens and/or a
T-cell response in the subject after administration of at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing. In some embodiments, the method may also
comprise continuing administration of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, if one or more neoantigens and/or a T-cell response is
detected.
[0054] Also provided herein are methods of treating a subject
having or suspected of having a neoplastic disorder, comprising
administering to the subject a therapeutically effective amount of
at least one compound chosen from at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing.
[0055] Also provided herein are neoantigen vaccines comprising at
least one neoantigen peptide. In some embodiments, the at least one
neoantigen peptide comprises a modified or novel neoantigen
sequence induced by contacting a neoplastic cell with a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing.
[0056] The methods and uses provided herein, in some embodiments,
may further comprise administering at least one additional therapy.
In some embodiments, the methods and uses provided herein may
result in lower systemic toxicity and/or improved tolerance.
[0057] Also disclosed herein is a method of treating cancer in a
subject in need thereof, comprising administering at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, and at least one additional therapy. Also
disclosed herein is a method of treating a subject having or
suspected of having a neoplastic disorder comprising administering
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, and at least one
additional therapy.
[0058] As described herein, compounds disclosure may be substituted
with one or more substituents, such as those illustrated generally
herein, or as exemplified by particular classes, subclasses, and
species of the disclosure. In general, the term "substituted"
refers to the replacement of hydrogen radicals in a given structure
with the radical of a specified substituent. Unless otherwise
indicated, a substituted group may have a substituent at each
substitutable position of the group, and when more than one
position in any given structure may be substituted with more than
one substituent chosen from a specified group, the substituent may
be either the same or different at every position. Combinations of
substituents envisioned by this disclosure are those that result in
the formation of stable or chemically feasible compounds.
[0059] "Stable" refers to compounds that are not substantially
altered chemically and/or physically when subjected to conditions
to allow for their production, detection, and their recovery,
purification, and use for one or more of the purposes disclosed
herein. In some embodiments, a stable compound or chemically
feasible compound is one that is not substantially altered when
kept at a temperature of 40.degree. C. or less, in the absence of
moisture or other chemically reactive conditions, for at least a
week.
[0060] "Isomers" refers to compounds having the same number and
kind of atoms, and hence the same molecular weight, but differing
with respect to the arrangement or configuration of the atoms.
"Stereoisomers" refers to compounds that have the same atomic
connectivity but different arrangements of their atoms in space.
"Diastereoisomers" or "diastereomers" refers to stereoisomers that
are not enantiomers. "Enantiomers" refers to stereoisomers that are
non-superimposable mirror images of one another.
[0061] Enantiomers taught herein may include "enantiomerically
pure" isomers that comprise substantially a single enantiomer, for
example, greater than or equal to 90%, 92%, 95%, 98%, or 99%, or
equal to 100% of a single enantiomer, at a particular asymmetric
center or centers. An "asymmetric center" or "chiral center" refers
to a tetrahedral carbon atom that comprises four different
substituents.
[0062] "Stereomerically pure" as used herein means a compound or
composition thereof that comprises one stereoisomer of a compound
and is substantially free of other stereoisomers of that compound.
For example, a stereomerically pure composition of a compound
having one chiral center will be substantially free of the opposite
enantiomer of the compound. In some embodiments, a stereomerically
pure composition of a compound having two chiral centers will be
substantially free of diastereomers, and substantially free of the
opposite enantiomer, of the compound. In some embodiments, a
stereomerically pure compound comprises greater than about 80% by
weight of one stereoisomer of the compound and less than about 20%
by weight of the other stereoisomers of the compound, such as
greater than about 90% by weight of one stereoisomer of the
compound and less than about 10% by weight of the other
stereoisomers of the compound, further such as greater than about
95% by weight of one stereoisomer of the compound and less than
about 5% by weight of the other stereoisomers of the compound, and
further such as greater than about 97% by weight of one
stereoisomer of the compound and less than about 3% by weight of
the other stereoisomers of the compound. See, e.g., U.S. Pat. No.
7,189,715.
[0063] "R" and "S" as terms describing isomers are descriptors of
the stereochemical configuration at an asymmetrically substituted
carbon atom. The designation of an asymmetrically substituted
carbon atom as "R" or "S" is done by application of the
Cahn-Ingold-Prelog priority rules, as are well known to those
skilled in the art, and described in the International Union of
Pure and Applied Chemistry (IUPAC) Rules for the Nomenclature of
Organic Chemistry. Section E, Stereochemistry.
[0064] "Amine oxide" or "amine-N-oxide" or "N-oxide" is a chemical
compound that contains the functional group
R.sup.3N.sup.+--P.sup.-, an N--O bond with three additional
hydrogen and/or hydrocarbon sidechains attached to N. Sometimes it
is written as R.sup.3N.fwdarw.O.
[0065] "Ar" or "aryl" refer to an aromatic carbocyclic moiety
having one or more closed rings. Examples include, without
limitation, phenyl, naphthyl, anthracenyl, phenanthracenyl,
biphenyl, and pyrenyl.
[0066] "Heteroaryl" refers to a cyclic moiety having one or more
closed rings, with one or more heteroatoms (oxygen, nitrogen or
sulfur) in at least one of the rings, wherein at least one of the
rings is aromatic, and wherein the ring or rings may independently
be fused, and/or bridged. Examples include without limitation
phenyl, thiophenyl, triazolyl, pyridinyl, pyrimidinyl, pyridazinyl,
and pyrazinyl.
[0067] "Alkyl" or "alkyl group," as used herein, means a
straight-chain (i.e., unbranched), branched, or cyclic hydrocarbon
chain that is completely saturated. In some embodiments, alkyl
groups contain 1-8 carbon atoms. In some embodiments, alkyl groups
contain 1-6 carbon atoms ("C.sub.1-C.sub.6 alkyl groups"). In some
embodiments, alkyl groups contain 1-3 carbon atoms. In still other
embodiments, alkyl groups contain 2-3 carbon atoms, and in some
embodiments, alkyl groups contain 1-2 carbon atoms. In some
embodiments, the term "alkyl" or "alkyl group" refers to a
cycloalkyl group, also known as carbocycle. Non-limiting examples
of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl,
cyclopropyl, and cyclohexyl.
[0068] "Alkoxy", as used herein, refers to an alkyl group, as
previously defined, attached to the principal carbon chain through
an oxygen ("alkoxy") atom.
[0069] "Haloalkyl" refers to an alkyl group substituted with one or
more halo atoms (F, Cl, Br, I). For example, "fluoromethyl" refers
to a methyl group substituted with one or more fluoro atoms (e.g.,
monofluoromethyl, difluoromethyl, or trifluoromethyl).
[0070] "Heteroatom" refers to O, S or N.
[0071] "Heterocyclyl" or "heterocyclic" as used herein, means a
monocyclic heterocycle, a bicyclic heterocycle, or a tricyclic
heterocycle containing at least one heteroatom in the ring.
[0072] The monocyclic heterocycle is a 3-, 4-, 5-, 6-, 7, or
8-membered ring containing at least one heteroatom independently
chosen from O, N, and S. In some embodiments, the heterocycle is a
3- or 4-membered ring containing one heteroatom chosen from O, N
and S. In some embodiments, the heterocycle is a 5-membered ring
containing zero or one double bond and one, two or three
heteroatoms chosen from O, N and S. In some embodiments, the
heterocycle is a 6-, 7-, or 8-membered ring containing zero, one or
two double bonds and one, two or three heteroatoms chosen from O, N
and S. Representative examples of monocyclic heterocycle include,
but are not limited to, azetidinyl, azepanyl, aziridinyl,
diazepanyl, 1 ,3-dioxanyl, 1,3-dioxolanyl, dihydropyranyl
(including 3,4-dihydro-2H-pyran-6-yl), 1,3-dithiolanyl,
1,3-dithianyl, imidazolinyl, imidazolidinyl, isothiazolinyl,
isothiazolidinyl, isoxazolinyl, isoxazolidinyl, morpholinyl,
oxadiazolinyl, oxadiazolidinyl, oxazolinyl, oxazolidinyl,
piperazinyl, piperidinyl, pyranyl, pyrazolinyl, pyrazolidinyl,
pyrrolinyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl
(including tetrahydro-2H-pyran-4-yl), tetrahydrothienyl,
thiadiazolinyl, thiadiazolidinyl, thiazolinyl, thiazolidinyl,
thiomorpholinyl, 1,1-dioxidothiomorpholinyl (thiomorpholine
sulfone), thiopyranyl, and trithianyl.
[0073] The bicyclic heterocycles of the present disclosure include
a monocyclic heterocycle fused to an aryl group, or a monocyclic
heterocycle fused to a monocyclic cycloalkyl, or a monocyclic
heterocycle fused to a monocyclic cycloalkenyl, or a monocyclic
heterocycle fused to a monocyclic heterocycle. Examples of bicyclic
heterocycles include, but are not limited to,
3,4-dihydro-2H-pyranyl, 1,3-benzodioxolyl, 1,3-benzodithiolyl,
2,3-dihydro-1,4-benzodioxinyl, 2,3-dihydro-1-benzofuranyl,
2,3-dihydro-1-benzothienyl, 2,3-dihydro-1H-indolyl, and
1,2,3,4-tetrahydroquinolinyl.
[0074] In some embodiments, the bicyclic heterocycle is a spiro
heterocycle. As known in the art, a "spiro" heterocycle is a
bicyclic moiety with rings connected through just one atom. The
connecting atom is also called the spiro atom and most often is a
quaternary atom such as carbon or nitrogen. Spiro compounds may be
designated with the infix spiro followed by square brackets
containing the number of atoms in the smaller ring and the number
of atoms in the larger ring excluding the spiroatom itself; the
numbers being separated by a dot. A non-limiting example of such
compounds is 2,6-diazaspiro[3.3]heptane.
[0075] The tricyclic heterocycle is a bicyclic heterocycle fused to
an aryl group, a bicyclic heterocycle fused to a monocyclic
cycloalkyl, a bicyclic heterocycle fused to a monocyclic
cycloalkenyl, or a bicyclic heterocycle fused to a monocyclic
heterocycle. Representative examples of tricyclic heterocycles
include, but are not limited to,
2,3,4,4a,9,9a-hexahydro-1H-carbazolyl,
5a,6,7,8,9,9a-hexahydrodibenzo[b,d]furanyl, and
5a,6,7,8,9,9a-hexahydrodibenzo[b, d]thienyl.
[0076] The heterocycle groups of the present disclosure are
connected to the parent molecular moiety through any substitutable
carbon atom or any substitutable nitrogen, oxygen or sulfur atom
contained within the groups and may contain one or two alkylene
bridges of 1, 2, 3, or 4 carbon atoms, each linking two
non-adjacent carbon atoms of the groups. Examples of such "bridged"
heterocycle groups include, but are not limited to,
oxatricyclo[3.3.1.1.sup.3,7]decyl (including 2-oxatricyclo
[3.3.1.].sup.3,7]decyl), 2,4-dioxabicyclo[4.2.1]nonyl,
oxabicyclo[2.2.1]heptyl (including 2-oxabicyclo[2.2.1]heptyl) and
2,5-diazabicyclo[2.2.1]heptane.
[0077] In the above heteroaryl and heterocycles the nitrogen or
sulfur atoms can be optionally oxidized to various oxidation
states. In a specific example, the group S(O).sub.0-2 refers to
--S--(sulfide), --S(O)-(sulfoxide), and --SO.sub.2-(sulfone)
respectively. For convenience, nitrogens, particularly but not
exclusively, those defined as annular aromatic nitrogens, are meant
to include those corresponding N-oxide forms. Thus, for a compound
of the disclosure having, for example, a pyridyl ring; the
corresponding pyridyl-N-oxide is meant to be included as another
compound of the disclosure.
[0078] "Treatment," "treat," or "treating" cancer refers to
reversing (e.g., overcoming a differentiation blockage of the
cells), alleviating (e.g., alleviating one or more symptoms, such
as fatigue from anemia, low blood counts, etc.), and/or delaying
the progression of (e.g., delaying the progression of the condition
such as transformation to AML) a cancer as described herein.
[0079] "Subject", as used herein, means an animal subject, such as
a mammalian subject, and particularly human beings.
[0080] The term "antibody" is used in the broadest sense to refer
to an immunoglobulin molecule that recognizes and specifically
binds to a target, such as a protein, polypeptide, carbohydrate,
polynucleotide, lipid, or combinations of the foregoing through at
least one antigen recognition site within the variable region of
the immunoglobulin molecule. The heavy chain of an antibody is
composed of a heavy chain variable domain (V.sub.H) and a heavy
chain constant region (C.sub.H). The light chain is composed of a
light chain variable domain (V.sub.L) and a light chain constant
domain (C.sub.L). For the purposes of this application, the mature
heavy chain and light chain variable domains each comprise three
complementarity determining regions (CDR1, CDR2 and CDR3) within
four framework regions (FR1, FR2, FR3, and FR4) arranged from
N-terminus to C-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
An "antibody" can be naturally occurring or man-made, such as
monoclonal antibodies produced by conventional hybridoma
technology. The term "antibody" includes full-length monoclonal
antibodies and full-length polyclonal antibodies, as well as
antibody fragments such as Fab, Fab', F(ab').sub.2, Fv, and single
chain antibodies. An antibody can be any one of the five major
classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or
subclasses thereof (e.g., isotypes IgG1, IgG2, IgG3, IgG4). The
term further encompasses human antibodies, chimeric antibodies,
humanized antibodies and any modified immunoglobulin molecule
containing an antigen recognition site, so long as it demonstrates
the desired biological activity (e.g., binds the target antigen,
internalizes within a target-antigen expressing cell).
[0081] "Pharmaceutically acceptable carrier" as used herein refers
to a nontoxic carrier, adjuvant, or vehicle that does not destroy
the pharmacological activity of the compound with which it is
formulated. Pharmaceutically acceptable carriers, adjuvants or
vehicles that may be used in the compositions of this disclosure
include, but are not limited to, ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin,
buffer substances such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts or electrolytes, such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances,
polyethylene glycol, cyclodextrins, sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
polyethylene glycol and wool fat.
[0082] A "pharmaceutically acceptable salt" is a salt that retains
the desired biological activity of the parent compound and does not
impart undesired toxicological effects. Examples of such salts are:
(a) acid addition salts formed with inorganic acids, for example,
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; and salts formed with organic
acids, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid,
[0083] alginic acid, polyglutamic acid, naphthalenesulfonic acid,
methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic
acid, polygalacturonic acid, and the like; and (b) salts formed
from elemental anions such as chlorine, bromine, and iodine. See,
e.g., Haynes et al., "Commentary: Occurrence of Pharmaceutically
Acceptable Anions and Cations in the Cambridge Structural
Database," J. Pharmaceutical Sciences, vol. 94, no. 10 (2005), and
Berge et al., "Pharmaceutical Salts", J. Pharmaceutical Sciences,
vol. 66, no. 1 (1977), which are incorporated by reference
herein.
[0084] Unless indicated otherwise, nomenclature used to describe
chemical groups or moieties as used herein follow the convention
where, reading the name from left to right, the point of attachment
to the rest of the molecule is at the right-hand side of the name.
For example, the group "(C.sub.1-3 alkoxy)C.sub.1-3 alkyl," is
attached to the rest of the molecule at the alkyl end. Further
examples include methoxyethyl, where the point of attachment is at
the ethyl end, and methylamino, where the point of attachment is at
the amine end.
[0085] Unless indicated otherwise, where a chemical group is
described by its chemical formula or structure having a terminal
bond moiety indicated by "--", it will be understood that the "--"
represents the point of attachment.
[0086] Unless otherwise stated, compounds depicted herein include
all enantiomeric, diastereomeric, and geometric (or conformational)
forms of the structure; for example, the R and S configurations for
each asymmetric center, (Z) and (E) double bond isomers, and (Z)
and (E) conformational isomers. Therefore, single stereochemical
isomers as well as enantiomeric, diastereomeric, and geometric (or
conformational) mixtures of the present compounds are within the
scope of the disclosure. Unless otherwise stated, all tautomeric
forms of the compounds of the disclosure are within the scope of
the disclosure. Additionally, unless otherwise stated, structures
depicted herein include compounds that differ only by the presence
of one or more isotopically enriched atoms. For example, compounds
having the formulae disclosed herein except for the replacement of
hydrogen by deuterium or tritium, or the replacement of a carbon by
a .sup.13C- or .sup.14C-enriched carbon are within the scope of
this disclosure. Such compounds may be useful, for example, as
analytical tools or probes in biological assays.
[0087] Provided herein according to some embodiments are compounds
of Formula I:
##STR00029##
and pharmaceutically acceptable salts thereof, [0088] wherein:
[0089] n is chosen from 0, 1, 2 or 3;
[0090] R.sup.1 is chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, --NR.sup.9R.sup.10 groups,
##STR00030##
groups,
##STR00031##
groups,
##STR00032##
groups,
##STR00033##
groups,
##STR00034##
groups,
##STR00035##
groups,
##STR00036##
groups, and
##STR00037##
groups;
[0091] R.sup.9 is chosen from hydrogen, --NR.sup.11R.sup.12 groups,
C.sub.1-C.sub.6 alkyl groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H
groups, C.sub.3-C.sub.8 cycloalkyl groups, and C.sub.3-C.sub.8
heterocyclyl groups, wherein the --NR.sup.11R.sup.12 groups,
C.sub.1-C.sub.6 alkyl groups, C.sub.3-C.sub.8 cycloalkyl groups,
and C.sub.3-C.sub.8 heterocyclyl groups may be unsubstituted or
substituted from 1-3 times with a group independently chosen from
C.sub.1-C.sub.6 alkyl groups, --(C.sub.1-C.sub.6 alkyl)--CO.sub.2H
groups, hydroxy, halogen groups, and C.sub.1-C.sub.6 alkoxy
groups;
[0092] R.sup.10 is chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups;
[0093] one of either R.sup.2 or R.sup.3 is chosen from hydrogen and
C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, --OR.sup.10, --OC(O)R.sup.10, --OC(O)R.sup.1, and
C.sub.1-C.sub.6 alkyl groups;
[0094] R.sup.4 is chosen from hydrogen and hydroxy;
[0095] R.sup.5 and R.sup.6 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups;
[0096] R.sup.7 and R.sup.8 are each independently chosen from
hydrogen, hydroxy, C.sub.1-C.sub.6 alkoxy groups, and
C.sub.1-C.sub.6 alkyl groups; and
[0097] Y is chosen from phenyl, thiophenyl, triazolyl, pyridinyl,
pyrimidinyl, pyridazinyl, and pyrazinyl, wherein Y may be
unsubstituted or substituted from 1-3 times with groups
independently chosen from oxo groups, C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.5 cycloalkyl groups, hydroxy C.sub.1-C.sub.6 alkyl
groups, C.sub.1-C.sub.6 alkoxy groups, methoxy C.sub.1-C.sub.6
alkyl groups, --NR.sup.11R.sup.12 groups,
##STR00038##
wherein R.sup.11 and R.sup.12 are each independently chosen from
hydrogen and C.sub.1-C.sub.6 alkyl groups.
[0098] In some embodiments, in Formula I, Y is
##STR00039##
[0099] In some embodiments, in Formula I, Y is chosen from
optionally substituted phenyl groups.
[0100] In some embodiments, in Formula I, Rl is chosen from
methyl,
##STR00040##
groups,
##STR00041##
groups,
##STR00042##
groups,
##STR00043##
groups,
##STR00044##
groups,
##STR00045##
groups,
##STR00046##
groups, and
##STR00047##
groups; wherein R.sup.9, R.sup.10, R.sup.11, and R.sup.12 are
defined as above.
[0101] Also provided herein are compounds of Formula II:
##STR00048##
and pharmaceutically acceptable salts thereof, [0102] wherein:
[0103] X is chosen from O, NR' groups, and CH.sub.2, wherein R' is
chosen from hydrogen and C.sub.1-C.sub.6 alkyl groups;
[0104] R.sup.1 is chosen from methyl, NR.sup.11R.sup.12 groups,
##STR00049##
groups, and
##STR00050##
groups;
[0105] R.sup.10 is chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, and halo C.sub.1-C.sub.6 alkyl
groups, wherein the C.sub.3-C.sub.8 cycloalkyl groups may be
unsubstituted or substituted from 1-3 times with a group
independently chosen from C.sub.1-C.sub.6 alkyl groups, hydroxy,
halogen groups, and C.sub.1-C.sub.6 alkoxy groups;
[0106] R.sup.11 and R.sup.12 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups;
[0107] one of either R.sup.2 or R.sup.3 is chosen from hydrogen and
C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, hydroxy and C.sub.1-C.sub.6 alkyl groups;
[0108] one of either R.sup.4 or R.sup.5 is hydrogen, and the other
is chosen from hydrogen, hydroxy, and
##STR00051##
[0109] R.sup.6 and R.sup.7 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups;
[0110] R.sup.8 and R.sup.9 are each independently chosen from
hydrogen and C.sub.1-C.sub.6 alkyl groups; or R.sup.8 and R.sup.9
are taken together to form a cyclopropyl ring; and
[0111] Y is chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, methoxy, and --NR.sup.13R.sup.14
groups, wherein R.sup.13 and R.sup.14 are each independently chosen
from hydrogen, C.sub.1-C.sub.6 alkyl groups, and methoxy
C.sub.1-C.sub.6 alkyl groups; or R.sup.13 and R.sup.14 may be taken
together with the N to form a group chosen from
##STR00052##
a morpholine, a piperidine, a thiazolidine, an indole, an indoline,
and an isoindoline ring;
[0112] wherein Y may be unsubstituted or substituted from 1-3 times
with a group independently chosen from C.sub.1-C.sub.6 alkyl
groups, hydroxy, hydroxy C.sub.1-C.sub.6 alkyl groups, methoxy,
methoxy C.sub.1-C.sub.6 alkyl groups, halo, halo C.sub.1-C.sub.6
alkyl groups, --C(O)NH.sub.2, --NHCOO--C.sub.1-C.sub.6 alkyl
groups, --COOH,
##STR00053##
and --NR.sup.15R.sup.16 groups, wherein R.sup.15 and R.sup.16 are
each independently chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups.
[0113] Also disclosed herein are compounds of Formula III:
##STR00054##
and pharmaceutically acceptable salts thereof, [0114] wherein:
[0115] n is chosen from 0, 1 and 2;
[0116] m is chosen from 1, 2, and 3;
[0117] R.sup.1 is chosen from C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups, --NR.sup.11R.sup.12 groups,
##STR00055##
groups,
##STR00056##
groups,
##STR00057##
groups,
##STR00058##
groups,
##STR00059##
groups,
##STR00060##
groups,
##STR00061##
groups,
##STR00062##
groups, and
##STR00063##
groups,
[0118] R.sup.11 is chosen from hydrogen, --NR.sup.16R.sup.17
groups, C.sub.1-C.sub.6 alkyl groups, --(C.sub.1-C.sub.6
alkyl)--CO.sub.2H groups, --(C.sub.1-C.sub.6
alkyl)--CO.sub.2R.sup.12 groups, --(C.sub.1-C.sub.6
alkyl)--NR.sup.16R.sup.17 groups, C.sub.3-C.sub.8 cycloalkyl
groups, and C.sub.3-C.sub.8 heterocyclyl groups, wherein the
--NR.sup.16R.sup.17 groups, C.sub.1-C.sub.6 alkyl groups,
C.sub.3-C.sub.8 cycloalkyl groups and C.sub.3-C.sub.8 heterocyclyl
groups may be unsubstituted or substituted from 1-3 times with a
group independently chosen from C.sub.1-C.sub.6 alkyl groups,
--(C.sub.1-C.sub.6 alkyl)--CO.sub.2H groups, hydroxy, halogen
groups, and C.sub.1-C.sub.6 alkoxy groups;
[0119] R.sup.12 is chosen from hydrogen and C.sub.1-C.sub.6 alkyl
groups;
[0120] one of either R.sup.2 or R.sup.3 is chosen from hydrogen and
C.sub.1-C.sub.6 alkyl groups, and the other is chosen from
hydrogen, --OR.sup.10, --OC(O)R.sup.10, --OC(O)R.sup.1, and
C.sub.1-C.sub.6 alkyl groups;
[0121] R.sup.4 is hydrogen or hydroxy;
[0122] R.sup.5 and R.sup.6 are each independently chosen from
C.sub.1-C.sub.6 alkyl groups;
[0123] R7 and R.sup.8 are each independently chosen from hydrogen,
hydroxy, C.sub.1-C.sub.6 alkoxy groups, and C.sub.1-C.sub.6 alkyl
groups; and
[0124] R.sup.9 and R.sup.10 are each independently chosen from
hydrogen, C.sub.1-C.sub.6 alkyl groups, hydroxy, and
C.sub.1-C.sub.6 alkoxy groups; or, one of R.sup.9 or R.sup.10 is
oxo and the other is absent;
[0125] Z is chosen from C.sub.1-C.sub.6 alkyl groups,
--C(O)--C.sub.1-C.sub.6 alkyl groups, --OR.sup.13, and
--NR.sup.14R.sup.15 groups,
[0126] wherein R.sup.13 is chosen from hydrogen, C.sub.1-C.sub.6
alkyl groups, and --C(O)--C.sub.1-C.sub.6 alkyl groups, wherein
R.sup.14 and R.sup.15 are each independently chosen from hydrogen,
C.sub.1-C.sub.6 alkyl groups, and methoxy C.sub.1-C.sub.6 alkyl
groups; or R.sup.14 and R.sup.15 may be taken together with the N
to form a group chosen from
##STR00064##
a morpholine, a piperidine, a thiazolidine, an indole, an indoline,
and an isoindoline ring;
[0127] wherein Z may be unsubstituted or substituted from 1-3 times
with a group independently chosen from C.sub.1-C.sub.6 alkyl
groups, C.sub.3-C.sub.5 cycloalkyl groups, hydroxy C.sub.1-C.sub.6
alkyl groups, C.sub.1-C.sub.6
[0128] alkoxy groups, methoxy C.sub.1-C.sub.6 alkyl groups,
--NR.sup.16R.sup.17 groups,
##STR00065##
wherein R.sup.16 and R.sup.17 are each independently chosen from
hydrogen and C.sub.1-C.sub.6 alkyl groups.
[0129] In some embodiments, in Formula III, R.sup.1 is chosen from
methyl,
##STR00066##
groups,
##STR00067##
groups,
##STR00068##
groups,
##STR00069##
groups,
##STR00070##
groups,
##STR00071##
groups,
##STR00072##
groups,
##STR00073##
groups, and
##STR00074##
groups, wherein R.sup.11, R.sup.12, R.sup.16, R.sup.17 are defined
as above.
[0130] Also disclosed herein are compounds chosen from:
##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079##
##STR00080## ##STR00081## ##STR00082## ##STR00083## ##STR00084##
##STR00085## ##STR00086## ##STR00087## ##STR00088## ##STR00089##
##STR00090## ##STR00091## ##STR00092## ##STR00093## ##STR00094##
##STR00095## ##STR00096## ##STR00097## ##STR00098## ##STR00099##
##STR00100## ##STR00101## ##STR00102## ##STR00103## ##STR00104##
##STR00105## ##STR00106## ##STR00107## ##STR00108## ##STR00109##
##STR00110## ##STR00111## ##STR00112## ##STR00113## ##STR00114##
##STR00115## ##STR00116##
and pharmaceutically acceptable salts thereof.
[0131] Also disclosed herein are compounds chosen from: [0132]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
9-oxo-9-pyrrolidin-1-ylona-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl-
]4-methylpiperazine-1-carboxylate; [0133]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-[[(2R,3R)-3-hydroxyp-
entan-2-yl]carbamoyloxy]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo--
1-oxacyclododec-4-en-6-yl] acetate; [0134]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-(propylcarbamoyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-y-
l] acetate; [0135]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-[methyl(propyl)carbamoyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec--
4-en-6-yl] acetate; [0136]
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
pyrrolidine-1-carboxylate; [0137]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-[methyl(propyl)carbamoyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec--
4-en-6-yl]4-cycloheptyl-4-oxidopiperazin-4-ium-1-carboxylate;
[0138]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(dimethylcarbamoyloxy)-6-methylhept-
a-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-
-yl] acetate; [0139]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-(diethylcarbamoyloxy)-6-methylhepta-
-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate; [0140] [(2S,3S,4E,6S,7S ,10S)-7,10-dihydroxy
-3,7-dimethyl-2-[(2E,4E,6
S)-6-methyl-7-[methyl(propan-2-yl)carbamoyl]
oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl] acetate;
[0141]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-[butyl(methyl)carbamoyl]oxy-6-methy-
lhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-6-yl] acetate; [0142]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-[butan-2-yl(methyl)carbamoyl]oxy-6--
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclodo-
dec-4-en-6-yl] acetate; [0143]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-carbamoyloxy-6-methylhepta-2,4-dien-
-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1
-oxacyclododec-4-en-6-yl] acetate; [0144] [(2S,3E,5E)-6-[(2S
,3S,4E,6S
,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-2-yl]-2-methylhepta-3,5 -dienyl]
(2R)-2-(methoxymethyl)pyrrolidine-1-carboxylate; [0145]
[(2S,3S,4E,6S,7S
,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-[2-(methoxyethylmethyl)carbamoyl]oxy-
-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-y-
l] acetate; [0146] [(2R,3E,5E)-6-[(2S,3S,4E,6S,7S
,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-
-2-yl]-2-methylhepta-3,5-dienyl] azetidine-1-carboxylate; [0147]
[(2R,3E,5E)-6-[(2S,3S,4E,6S
,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-2-yl]-2-methylhepta-3,5 -dienyl]
(2S)-2-methylpyrrolidine-1-carboxylate; [0148]
[(2R,3E,5E)-6-[(2S,3S,4E,6S
,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-2-yl]-2-methylhepta-3,5-dienyl]
(2S)-2-methylpyrrolidine-1-carboxylate; [0149]
[(2R,3E,5E)-6-[(2S,3S,4E,6S
,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-2-yl]-2-methylhepta-3,5-dienyl] piperidine-1-carboxylate;
[0150] [(2R,3E,5E)-6-[(2S,3S,4E,6S
,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-2-yl]-2-methylhepta-3,5-dienyl]
(2R)-2-(hydroxymethyl)pyrrolidine-1-carboxylate; [0151]
[(2R,3E,5E)-6-[(2S,3S,4E,6S
,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-2-yl]-2-methylhepta-3,5-dienyl]
(3R)-3-hydroxypyrrolidine-1-carboxylate; [0152]
[(2R,3E,5E)-6-[(2S,3S,4E,6S ,7
S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-e-
n-2-yl]-2-methylhepta-3,5-dienyl] morpholine-4-carboxylate; [0153]
[(2R,3E,5E)-6-[(2S,3S,4E,6S
,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-2-yl]-2-methylhepta-3,5-dienyl]
4-methylpiperazine-1-carboxylate; 3-thiazolidinecarboxylic acid
[(2R,3E,5E)-6-[(2R,3S,4E,6R,7R,10R)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
ester; [0154]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6--
methyl-7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclo-
dodec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0155]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrro-
lidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-o-
xacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0156]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymeth-
yl)pyrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-
-oxo-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[0157]
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-6-(4-meth-
ylpiperazine-1-carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhept-
a-3,5-dienyl]1,3-dihydroisoindole-2-carboxylate; [0158]
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-6-(4-meth-
ylpiperazine-1-carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhept-
a-3,5-dienyl] indole-1-carboxylate; [0159]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-[2-(1-hydroxyethyl)p-
yrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-
-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[0160]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,2-dimethylpyrrolidine-1-carbonyl-
)oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxa-
cyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0161]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2S,5S)-2,5-dimethylpyrrolidine-1--
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-o-
xo-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[0162]
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-6-(4-meth-
ylpiperazine-1-carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhept-
a-3,5-dienyl] 2,3-dihydroindole-1-carboxylate; [0163]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-fluoropyrrolidine-1-carbony-
l]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0164]
[(2S,3S,4E,6S,7S
,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-
-oxo-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[0165]
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-6-(4-meth-
ylpiperazine-1-carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhept-
a-3,5-dienyl] 2-oxa-5-azaspiro[3.4] octane-5-carboxylate; [0166]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
acetate; [0167]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E-
,6S)-6-pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
acetate; [0168] [(2S,3S,4E,6S,7S
,10S)-7,10-dihydroxy-2-[(2E,4E)-6-[6-[(2R)-1-hydroxypropan-2-yl]pyridin-2-
-yl]hepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0169]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E)-6-[2-(dimethylamino)pyrimidin-4-yl]hepta-
-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] 4-methylpiperazine-1-carboxylate; [0170]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacydodec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0171]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idazin-3-ylhepta-2,4-dien-2-yl]-1-oxacydodec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0172]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
imidin-2-ylhepta-2,4-dien-2-yl]-1-oxacydodec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0173]
[(2R,3R,4E,6S,7R,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6R)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-propan-2-ylpiperazine-1-carboxylate; [0174]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-tert-butylpiperazine-1-carboxylate; [0175]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacydodec-4-en-6-yl]
4-cyclopentylpiperazine-1-carboxylate; [0176]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(oxan-4-yl)piperazine-1-carboxylate; [0177]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
6-cycloheptyl-2,6-diazaspiro[3.3]heptane-2-carboxylate; [0178]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacydodec-4-en-6-yl]
4-cycloheptyl-3-methylpiperazine-1-carboxylate; [0179]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cyclobutylpiperazine-1-carboxylate; [0180]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
N-methyl-N-(1-methylpiperidin-4-yl)carbamate; [0181]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
morpholine-4-carboxylate; [0182]
[(2R,3R,4E,6S,7R,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6R)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] (1S
,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate; [0183]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
8-cycloheptyl-3,8-diazabicyclo[3.2.1]octane-3-carboxylate; [0184]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methyl-1,4-diazepane-1-carboxylate; [0185]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cyclohexylpiperazine-1-carboxylate; [0186]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
piperazine-1-carboxylate; [0187]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptyl-1,4-diazepane-1-carboxylate; [0188]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-hydroxy-6-methylhept-
a-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-y]
4-methylpiperazine-1-carboxylate; [0189]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-y]
4-(azepan-1-yl)piperidine-1-carboxylate; [0190]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(8,8-difluoro-3-azabicyclo[3.2.1]octan-3-yl)piperidine-1-carboxylate;
[0191]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6--
methyl-9-oxo-9-pyrrolidin-1-ylnona-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0192]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6S)-6-methyl--
7-[methyl(propyl)carbamoyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec--
4-en-6-yl]4-cycloheptylpiperazine-1-carboxylate; [0193]
[(2S,3S,4E,6S,7S
,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-
-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0194]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrro-
lidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-o-
xacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[0195] [(2S,3S,4E,6S,7S
,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclod-
odec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0196]
[(2S,3S,4E,6S,7S,10R)-7-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(py-
rrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-10-(pyrrolidine-1-carb-
onyloxy)-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0197] [(2S,3S,4E,6S,7S
,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(2S)-2-methyl-
pyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-
-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0198]
[(2S,3S,4E,6S,7S
,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(3R)-3-methyl-
pyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-
-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0199]
[(2S,3S,4E,6S,7S
,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(3R)-3-methyl-
pyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-
-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0200]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2R)-2-carbamoylpyrrolidine-1-carb-
onyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
-oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[0201] [(2S,3S,4E,6S,7S
,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-[(2R)-2-(methoxymethyl)pyrrolidine-1-
-carbonyl]oxy
-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] 4-cycloheptylpiperazine-1-carboxylate; [0202] [(2S,3S,4E,6S,7S
,10S)-2-[(2E,4E,6R)-7-[(2S
,5S)-2,5-dimethylpyrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]--
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0203]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-fluoropyrrolidine-1-carbony-
l]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[0204]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-fluoropyrrolidine-1-carbony-
l]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[0205]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,2-dimethylpyrrolidine-1-carbonyl-
)oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxa-
cyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0206]
[(2S,3S,4E,6R,7R,10R)-2-[(2E,4E)-6,6-dimethyl-7-(pyrrolidine-1-carbonylox-
y)hepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec--
4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0207]
[(2S,3S,4E,6R,7R,10S)-2-[(2E,4E)-6,6-dimethyl-7-(pyrrolidine-1-carbonylox-
y)hepta-2,4-dien-2-yl]-7-hydroxy-3,7-dimethyl-12-oxo-10-(pyrrolidine-1-car-
bonyloxy)-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0208]
(2R)-1-[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-cycloheptylpiperazine-1-c-
arbonyl)oxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-2-yl]--
2-methylhepta-3,5-dienoxy]carbonylpyrrolidine-2-carboxylic
acid;
[0209]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6--
methyl-7-(3-oxopyrrolidine-1-carbonyl)oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxa-
cyclododec-4-en-6-yl]4-cycloheptylpiperazine-1-carboxylate; [0210]
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-cycloheptylpiperazine-1-carbonyl-
)oxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methy-
lhepta-3,5-dienyl]2-oxa-7-azaspiro[3.4]octane-7-carboxylate; [0211]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl]4-cycloheptylpiperazine-1-carboxylate; [0212]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-5-[1--
(pyrrolidine-1-carbonyloxymethyl)cyclopropyl]penta-2,4-dien-2-yl]-1-oxacyc-
lododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0213]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3S,4R)-3,4-dihydroxypyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12--
oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0214]
(3S)-1-[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-cycloheptylpiperaz-
ine-1-carbonyl)oxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-
-2-yl]-2-methylhepta-3,5-dienoxy]carbonylpyrrolidine-3-carboxylic
acid; [0215]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3S)-3-(dimethylamino)pyrro-
lidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dime-
thyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0216]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,5-dihydropyrrole-1-carbonyloxy)--
6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclo-
dodec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0217]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12--
oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0218]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6--
methyl-7-[(3S)-3-[(2-methylpropan-2-yl)oxycarbonylamino]pyrrolidine-1-carb-
onyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0219]
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-cycloheptylpiperazine-1-carbonyl-
)oxy-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methy-
lhepta-3,5-dienyl]3-azabicyclo[3.1.0]hexane-3-carboxylate; [0220]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0221]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0222]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-2-ylhexa-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0223]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-(2--
pyrrolidin-1-ylpyrimidin-4-yl)hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6--
yl] 4-cycloheptylpiperazine-1-carboxylate; [0224]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
azin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0225]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E)-6-[2-(dimethylamino)pyrimidin-4-yl]hepta-
-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] 4-cycloheptylpiperazine-1-carboxylate; [0226]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-(3-methylp-
yridin-2-yl)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0227]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-(4-methylp-
yridin-2-yl)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0228]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
imidin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0229]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idazin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0230]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
imidin-4-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0231]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyrimidin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0232]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyrimidin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0233]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-(4-methylp-
yrimidin-2-yl)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0234]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-(6--
pyrrolidin-1-ylpyridin-2-yl)hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl-
] 4-cycloheptylpiperazine-1-carboxylate; [0235]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(p-
yrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en--
6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0236]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidi-
ne-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacy-
clododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0237]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(p-
yrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en--
6-yl] 4-methylpiperazine-1-carboxylate; [0238]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidi-
ne-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacy-
clododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0239]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)p-
yrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-
-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[0240]
[(2S,3S,4E,6R,7R,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-10-hydroxy-3,7-dimethyl-12-oxo--
1-oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate;
[0241]
[(2S,3S,4E,6R,7R,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-10-hydroxy-3,7-dimethyl-12-oxo--
1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0242]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyri-
din-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate;
[0243]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyri-
din-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0244]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyri-
din-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0245]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyri-
din-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
N,N-dimethylcarbamate; [0246]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy
-7-[(2R,3R)-3-[(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,-
4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0247]
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-ca-
rbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0248]
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-carbonylo-
xy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0249]
[(2R,3E,5E)-6-[(2S,3S,4E,6R)-6-(dimethylcarbamoyloxy)-3-methyl-12-oxo-1-o-
xacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
pyrrolidine-1-carboxylate; [0250]
[(2R,3E,5E)-6-[(2S,3S,4E,6R)-6-(dimethylcarbamoyloxy)-3-methyl-12-oxo-1-o-
xacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(3R)-3-hydroxypyrrolidine-1-carboxylate; [0251]
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-carbonyl]oxy--
6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0252]
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-[(2S)-2-methylpyrrolidin-
e-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0253]
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-1-carbo-
nyl]oxy-6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en--
6-yl] 4-methylpiperazine-1-carboxylate; [0254]
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-carbonyl]oxy--
6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0255]
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-1-carbo-
nyl]oxy-6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en--
6-yl] 4-(2,2,2-trifluoroethyl)piperazine-1-carboxylate; [0256]
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-
-2-yl]-1-oxacyclododec-4-en-6-yl] acetate; [0257]
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-
-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0258]
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-ylhepta-2-
,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0259]
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-ylhepta-2-
,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] N,N-dimethylcarbamate;
[0260]
[(2S,3S,4E,6R)-2-[(2E,4E)-6-[2-(dimethylamino)pyrimidin-4-yl]hepta-2,4-di-
en-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0261]
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-(2-pyrrolidin-1-ylpyrimidin-4-
-yl)hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0262]
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-[2-[(3S)-3-triethylsilyloxypy-
rrolidin-1-yl]pyrimidin-4-yl]hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-y-
l] 4-cycloheptylpiperazine-1-carboxylate; [0263]
[(2S,3S,4E,6R)-2-[(2E,4E)-6-[2-[(3R)-3-hydroxypyrrolidin-1-yl]pyrimidin-4-
-yl]hepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0264]
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-pyrimidin-2-ylhepta-2,4-dien--
2-yl]-1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate;
[0265]
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrol-
idine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0266]
[(2S,3S,4E,6S,7S)-7-hydroxy-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrol-
idine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-ox-
acyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0267]
[(2S,3S,4E,6S,7S)-7-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1--
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclodo-
dec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0268]
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(3S)-3-
-(1-phenyltetrazol-5)oxypyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]
-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0269]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6--
methyl-7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclo-
dodec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0270]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-methylpiperazine-1-carboxylate; [0271]
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0272]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-methylpiperazine-1-carboxylate; [0273]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-(pyrrolidine-1-carbonylamino)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-
-4-en-6-yl] 4-cycloheptylpiperazine-1-carb oxylate; [0274]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-[[(2R)-2-(hydroxymet-
hyl)pyrrolidine-1-carbonyl]amino]-6-methylhepta-2,4-dien-2-yl]-3,7-dimethy-
l-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0275]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6--
methyl-7-(pyrrolidine-1-carbonylamino)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyc-
lododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0276]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl--
7-[methyl(pyrrolidine-1-carbonyl)amino]hepta-2,4-dien-2-yl]-12-oxo-1-oxacy-
clododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0277]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(4-cyclopropyltriazol-1-yl)-6-methy-
lhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0278]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6S)-7-methoxycarbonyloxy-6-
-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0279]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-9-methoxy-6-methyl-9-o-
xonona-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0280]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(cyclopentanecarbonylamino)-6-methy-
lhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate; [0281]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(cyclopentanecarbonylamino)-6-methy-
lhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
-en-6-yl]4-methylpiperazine-1-carboxylate; [0282]
4-cycloheptyl-1-piperazinecarboxylic acid
[(2R,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-7-[ox-
o(1-pyrrolidinyl)methoxy]hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
ester; [0283]
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidi-
ne-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacy-
clododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate; [0284]
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-carbonyl]oxy--
6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-azacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0285]
[(2R,3E,5E)-2-methyl-6-[(2S,3S,4E,6R)-3-methyl-6-[(4-methylpiperazine-1-c-
arbonyl)amino]-12-oxo-1-oxacyclododec-4-en-2-yl]hepta-3,5-dienyl]
pyrrolidine-1-carboxylate; [0286]
[(2S,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-7,10-dihydroxy-3,7-dimeth-
yl-12-oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
(2R,3R)-3-hydroxy-2-methylpentanoate; [0287]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E,6R)-7-hydroxy-6-methylhept-
a-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate;
[0288]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E-
)-6-pyridin-4-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0289]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0290]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0291]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-7-methyl-6-p-
yridin-2-ylocta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0292]
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-[(2R,3R)-3-[(2R,3R)-3-acetyl-
oxypentan-2-yl]oxiran-2-yl]-6-hydroxy-6-methylhepta-2,4-dien-2-yl]-7,10-di-
hydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0293]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E)-6-hydroxy-6-methyl-8-phen-
ylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0294]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E)-6-hydroxy-6-phenylhepta-2-
,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0295]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E)-6-hydroxy-6-thioph-
en-2-ylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0296]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-phe-
nylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate; [0297]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-6-(6-methoxypyridin-2-yl)-
hepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0298]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-[6-(2-meth-
ylpropoxy)pyridin-2-yl]hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate; [0299]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-methyl-8-p-
yridin-2-ylocta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0300]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-met-
hyl-7-pyridin-2-ylhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0301]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E,6R)-6-hydroxy-6-methyl-8-p-
henylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
acetate; [0302]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-2-ylhexa-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate;
[0303]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-3-ylhexa-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate;
[0304]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-4-ylhexa-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate;
[0305]
[(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-2-[(2E,4E)-6-hydroxy-8-(4-hydroxyphe-
nyl)-6-methylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en--
6-yl] acetate; [0306]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E)-6-methyl-8-p-
henylocta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl] acetate;
[0307]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-8-[2-(methoxymethyl)pheny-
l]-6-methylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate; [0308]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-8-[4-(methoxymethyl)pheny-
l]-6-methylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate; [0309]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-8-[3-(methoxymethyl)pheny-
l]-6-methylocta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6--
yl] acetate; [0310]
[(2S,3S,4E,6S,7S)-7-hydroxy-2-[(2E,4E,6S)-6-hydroxy-6-methyl-7-[(2R,3R)-3-
-[(2S)-3-oxopentan-2-yl]oxiran-2-yl]hepta-2,4-dien-2-yl]-3,7-dimethyl-10,1-
2-dioxo-1-oxacyclododec-4-en-6-yl] acetate; [0311]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6E,8S)-
-8-pyridin-2-ylnona-2,4,6-trien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate; [0312]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E,6S)-6--
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methyl-4-oxidopiperazin-4-ium-1-carboxylate; [0313]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(4-fluoropiperidin-1-yl)piperidine-1-carboxylate; [0314]
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-12-oxo-2-[(2E,4E)-6-pyr-
idin-3-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(4,4-difluoropiperidin-1-yl)piperidine-1-carboxylate; [0315]
(4S,7S,8S,9E,11S,12S)-4,7,8-trihydroxy-7,11-dimethyl-12-[(2E,4E,6S)-6-pyr-
idin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-9-en-2-one; [0316]
[(2S,3S,4E,6S,7S,10S)-7-acetyloxy-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(-
2R,3R)-3-[(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-
-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
piperazine-1-carboxylate; [0317]
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
piperazine-1-carboxylate; [0318] (2S,3S ,6S,7R,10R,E)-7-acetoxy
-10-hydroxy-2-((S,2E,4E)-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxira-
n-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-
-6-yl piperazine-1-carboxylate; [0319]
[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(-
2R,3R)-3-[(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-
-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-7-yl]
piperazine-1-carboxylate; [0320]
[(2S,3S,4E,6S,7S,10S)-7-acetyloxy-10-hydroxy-2-[(2E,4E,6R)-7-[(2R,3R)-3-[-
(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-
-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
piperazine-1-carboxylate; [0321]
[(2S,3S,4E,6S,7R,10R)-7-ethoxy-10-hydroxy-2-[(2E,4E,6R)-6-hydroxy--
7-[(2R,3R)-3-[(2S,3S)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4--
dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0322]
[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-10-hydroxy-2-[(2E,4E,6R)-7-[(2R,3R)-3-[-
(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,7-
-dimethyl-12-oxo-1-oxacyclododec-4-en-7-yl]
piperazine-1-carboxylate; [0323]
[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(2R,3R)-3-[(2R,3-
R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-methox-
y-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
piperazine-1-carboxylate; [0324] [(2
S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(2R,3R)-3-[(2R,3R)-3-hydro-
xypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-methoxy-3,7-dime-
thyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate; [0325]
[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R-
)-3-[(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl-
]-7-methoxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
N-methyl-N[2-(methylamino)ethyl]carbamate; [0326]
[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-3-[(2-
R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-met-
hoxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
N-methyl-N[2-(dimethylamino)ethyl]carbamate; [0327]
3-[4-[[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-
-3-[(2R,3R)-3-
hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-methoxy-3,-
7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]oxycarbonyl]piperazin-2-yl]pro-
panoic acid; [0328]
4-[4-[[(2S,3S,4E,6S,7S,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-
-3-[(2R,3R)-3-
hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-7-methoxy-3,-
7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]oxycarbonyl]piperazin-1-yl]but-
anoic acid; [0329] (2S,3S,6S,7R,10R,E)-7-acetoxy
-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan--
2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclo-
dodec-4-en-6-yl
(1S,4S)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate; [0330]
(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7--
((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)
oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclodode-
c-4-en-7-yl 2,5-diazabicyclo[2.2.1]heptane-2-carboxylate; [0331]
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
4-propylpiperazine-1-carboxylate; [0332] (2R,3 S,6S
,7R,10R,E)-6-acetoxy-10-hydroxy-2-((2S,6R,E)-6-hydroxy-7-((2R,3R)-3-((2R,-
3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhept-4-en-2-yl)-3,7-dimethyl-
-12-oxooxacyclododec-4-en-7-yl
4-(2-hydroxyethyl)piperazine-1-carboxylate; [0333]
(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7--
((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-di-
en-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-yl
4-methylpiperazine-1-carboxylate; [0334]
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
4-(2-aminoethyl)piperazine-1-carboxylate; [0335]
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
4-(2-ethoxy-2-oxoethyl)piperazine-1-carboxylate; [0336]
(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((-
2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7--
dimethyl-12-oxooxacyclododec-4-en-6-yl4-methylpiperazine-1-carboxylate;
[0337]
(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3-
R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-y-
l)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
piperazine-1-carboxylate; [0338] and pharmaceutically acceptable
salts thereof.
[0339] Disclosed herein are compositions comprising at least one
compound of the present disclosure (e.g., compounds of Formulas I,
II, and III) and/or pharmaceutically acceptable salts thereof and
at least one pharmaceutically acceptable carrier. The at least one
pharmaceutically acceptable carrier may be chosen according to the
particular route of administration for which the composition is
intended.
[0340] The pharmaceutical compositions of the present disclosure
may be formulated for parenteral, oral, inhalation spray, topical,
rectal, nasal, buccal, vaginal or implanted reservoir
administration, etc. The term "parenteral" as used herein includes
subcutaneous, intravenous, intramuscular, intra-articular,
intra-synovial, intrasternal, intrathecal, intrahepatic,
intralesional and intracranial injection or infusion techniques. In
some embodiments, the compositions are administered intravenously,
orally, subcutaneously, or via intramuscular administration.
Sterile injectable forms of the compositions of this disclosure may
be aqueous or oleaginous suspension. These suspensions may be
formulated according to techniques known in the art using suitable
dispersing or wetting agents and suspending agents. The sterile
injectable preparation may also be a sterile injectable solution or
suspension in a nontoxic parenterally acceptable diluent or
solvent, for example as a solution in 1,3-butanediol. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium.
[0341] Any bland fixed oil may be employed including synthetic
mono- or di-glycerides. Fatty acids, such as oleic acid and its
glyceride derivatives are useful in the preparation of injectables,
as are natural pharmaceutically acceptable oils, such as olive oil
or castor oil, especially in their polyoxyethylated versions. These
oil solutions or suspensions may also contain a long-chain alcohol
diluent or dispersant, such as carboxymethyl cellulose or similar
dispersing agents that are commonly used in the formulation of
pharmaceutically acceptable dosage forms including emulsions and
suspensions. Other commonly used surfactants, such as Tweens, Spans
and other emulsifying agents or bioavailability enhancers which are
commonly used in the manufacture of pharmaceutically acceptable
solid, liquid, or other dosage forms, may also be used for the
purposes of formulation.
[0342] For oral administration, a compound of the present
disclosure (e.g., Formulas I, II, or III) and/or a pharmaceutically
acceptable salt thereof may be provided in an acceptable oral
dosage form, including, but not limited to, capsules, tablets,
aqueous suspensions or solutions. In the case of tablets for oral
use, carriers commonly used include lactose and corn starch.
Lubricating agents, such as magnesium stearate, may also be added.
For oral administration in a capsule form, useful diluents include
lactose and dried cornstarch. When aqueous suspensions are required
for oral use, the active ingredient may be combined with an
emulsifying and/or suspending agent. If desired, certain
sweetening, flavoring or coloring agents may also be added.
[0343] Compounds and compositions of the present disclosure may be
used to treat various types of cancers, including those responsive
to agents that target the spliceosome, including SF3B1. As noted
above, the anti-tumor activity of pladienolide B is reported as
being connected to its targeting of the SF3b complex, inhibiting
splicing and altering the pattern of gene expression (Kotake et
al., "Splicing factor SF3b as a target of the antitumor natural
product pladienolide," Nature Chemical Biology 2007, 3, 570-575).
Mutations in the Splicing factor 3B subunit 1 (SF3B1) protein are
known to be implicated in a number of cancers, such as hematologic
malignancies and solid tumors. Scott et al., "Acquired mutations
that affect pre-mRNA splicing in hematologic malignancies and solid
tumors," JNCI 105, 20, 1540-1549.
[0344] Accordingly, the compounds (e.g., compounds of Formulas I,
II, and III and pharmaceutically acceptable salts of the foregoing)
and compositions of the present disclosure may be used to treat
hematological malignancies, such as, for example, cancers of the
blood (leukemia) and cancers of the lymph nodes (lymphomas).
Leukemias include acute lymphoblastic leukemia (ALL), acute
myleogenous leukemia (AML), chronic lymphocytic leukemia (CLL),
chronic myelogenous leukemia (CML), chronic myelomonocytic leukemia
(CMML), acute monocytic leukemia (AMoL), etc. Lymphomas include
Hodgkin's lymphoma and non-Hodgkin's lymphoma. Other hematologic
malignancies may include myelodysplastic syndrome (MDS).
[0345] Solid tumors include carcinomas such as adenocarcinoma,
e.g., breast cancer, pancreatic cancer, prostate cancer, colon or
colorectal cancer, lung cancer, gastric cancer, cervical cancer,
endometrial cancer, ovarian cancer, cholangiocarcinoma, glioma,
melanoma, etc.
[0346] The compounds (e.g., compounds of Formulas I, II, and III)
and pharmaceutically acceptable salts thereof and compositions of
the present disclosure may also be used to treat cancers that may
be responsive to agents that target a spliceosome gene or protein
other than SF3B1. The following are non-limiting examples of
cancers responsive to agents that target the spliceosome. Thus,
compounds of the present disclosure may be administered to subjects
to treat a variety of such cancers or conditions, particularly
patients or subjects afflicted with:
[0347] a) Myelodysplastic syndrome (MDS): See, e.g., "SF3B1
mutations in myelodysplastic syndromes: clinical associations and
prognostic implications," Damm F. et al. Leukemia, 2011, 1-4;
"Frequent pathway mutations in splicing machinery in
myelodysplasia," Yoshida K. et al, Nature, 2011, 478, 64-69;
"Clinical significance of SF3B1 mutations in myelodysplastic
syndromes and myelodysplastic/myeloproliferative neoplasms,"
Malcovati L. et al., Blood, 2011, 118, 24, 6239-6246; "Mutations in
the spliceosome machinery, a novel and ubiquitous pathway in
leukemogenesis," Makishima et al, Blood, 2012, 119, 3203-3210;
"Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts,"
Pappaemannuil, E. et al, New England J. Med. 2011, DOI
10.1056/NEJMoa1103283.
[0348] b) Chronic lymphocytic leukemia (CLL): See, e.g., "Defects
in the spliceosomal machinery: a new pathway of leukaemogenesis,"
Maciejewski, J. P., Padgett, R. A., Br. J. Haematology, 2012, 1-9;
"Mutations in the SF3B1 splicing factor in chronic lymphocytic
leukemia: associations with progression and
fludarabine-refractoriness," Rossi et al, Blood, 2011, 118,
6904-6908; "Exome sequencing identifies recurrent mutations of the
splicing factor SF3B1 gene in chronic lymphocytic leukemia,"
Quesada et al, Nature Genetics, 2011, 44, 47-52.
[0349] c) Chronic myelomonocytic leukemia (CMML): See, e.g.,
Yoshida et al, Nature 2011; "Spliceosomal gene mutations are
frequent events in the diverse mutational spectrum of chronic
myelomonocytic leukemia but largely absent in juvenile
myelomonocytic leukemia," Kar S. A. et al, Haematologia, 2012, DOI:
10.3324/haemato1.2012.064048.
[0350] d) Acute myeloid leukemia (AML): See, e.g., Malcovati et
al., Blood 2011; Yoshida et al, Nature 2011.
[0351] e) Breast cancer: See, e.g., "Whole genome analysis informs
breast cancer response to aromatase inhibition," Ellis et al,
Nature, 2012, 486, 353-360.
[0352] f) Uveal melanoma.sup.. See, e.g., "SF3B1 mutations are
associated with alternative splicing in uveal melanoma", Furney et
al, Cancer Disc. 2013, 10, 1122-1129.
[0353] g) Endometrial cancer: See, e.g., Tefferi et al.,
"Myelodysplastic syndromes." N Engl J Med. 2009; 361:1872-85.
[0354] h) Gastric cancer: See, e.g., Int J Cancer. 2013
July;133(1):260-5, "Mutational analysis of splicing machinery genes
SF3B1, U2AF1 and SRSF2 in myelodysplasia and other common tumors."
Je et al.
[0355] i) Ovarian cancer: See, e.g., Int J Cancer. 2013
July;133(1):260-5, "Mutational analysis of splicing machinery genes
SF3B1, U2AF1 and SRSF2 in myelodysplasia and other common tumors."
Je et al.
[0356] j) Biliary Tract cancers such as Cholangiocarcinoma and
Pancreatic cancer: See, e.g., Biankin et al., "Pancreatic cancer
genomes reveal aberrations in axon guidance pathway genes," Nature
2012, 491, 399-405.
[0357] k) Lung cancer: See, e.g., "Exome sequencing identifies
recurrent mutations of the splicing factor SF3B1 gene in chronic
lymphocytic leukemia," Quesada et al., Nature Genetics 44, 47-52
(2012); Scott et al., "Acquired mutations that affect pre-mRNA
splicing in hematologic malignancies and solid tumors," JNCI 105,
20, 1540-1549.
[0358] In addition, the Catalogue of somatic mutations in cancer
(COSMIC) (Wellcome Trust Sanger Institute, Genome Research Limited,
England) reports SF3B1 mutations have been found in various types
of cancer samples.
[0359] A compound of the present disclosure (e.g., a compound of
Formulas I, II, or III) may be administered to a subject in a
treatment effective or therapeutically effective amount. The amount
of a compound of the present disclosure that may be combined with a
carrier material to produce a composition in a single dosage form
will vary depending upon the subject treated and the particular
route of administration. In some embodiments, a dose of 0.01
mg/kg-100 mg/kg body weight/day of the at least one compound
disclosed herein is administered. In some embodiments, the dose is
from from 0.01 mg to 50 mg of the at least one compound disclosed
herein. In some embodiments, 0.1 mg to 25 mg of the at least one
compound disclosed herein is provided. In some embodiments, 5 mg to
40 mg of the at least compound disclosed herein is provided.
[0360] One of ordinary skill will understand that a specific dosage
and treatment regimen for a particular patient will depend upon a
variety of factors, including the activity of the specific compound
employed, the age, body weight, general health, sex, diet, time of
administration, rate of excretion, drug combination, the judgment
of the treating physician, and the severity of the particular
disease being treated. The amount of the the at least one compound
disclosed herein will also depend upon the particular compound/salt
being used.
[0361] In some embodiments, the cancer is tested for and/or is
positive for one or more mutations in the Splicing factor 3B
subunit 1 (SF3B1) gene or protein, wherein the presence of the
mutation(s) ("positive") indicates the subject's cancer is
responsive to a method of treatment comprising administration of
the at least one compound disclosed herein targeting this protein
and/or the spliceosome. Examples of such spliceosome genes include,
but are not limited to, those presented in Table 1
TABLE-US-00001 TABLE 1 Spliceosome genes and potential diseases
affected Spliceosome gene Disease(s) Splicing factor 3B subunit 1
(SF3B1) see listings above U2 small nuclear RNA auxiliary factor 1
MDS, AML, CMML, LUAD, UCEC (U2AF1) CMML, MDS, PMF, AML
Serine/arginine-rich splicing factor 2 (SRSF2) MDS Zinc finger
(CCCH type), RNA-binding Retinitis Pigmentosa motif and
serine/arginine rich 2 (ZRSR2) Pre-mRNA-processing-splicing factor
8 (PRPF8) Myeloid neoplasms U2 Small Nuclear RNA Auxiliary Factor 2
MDS, PRAD, (U2AF2) COAD Splicing Factor 1 (SF1) myeloid neoplasms,
OV, COAD Splicing factor 3a subunit 1 (SF3A1) MDS PRP40 pre-mRNA
processing factor 40 homolog B LUAD (PRPF40B) RNA Binding Motif
Protein 10 (RBM10) COAD Poly(rC) binding protein 1 (PCBP1) SKCM
Crooked neck pre-mRNA splicing factor 1 LUSC (CRNKL1) DEAH
(Asp-Glu-Ala-His) box helicase 9 (DHX9) STAD Peptidyl-prolyl
cis-trans isomerase-like 2 (PPIL2) SKCM RNA binding motif protein
22 (RBM22) LUAD Small nuclear ribonucleoprotein Sm D3 (SNRPD3) GBM,
LGG Probable ATP-dependent RNA helicase DDX5 LUAD (DDX5)
Pre-mRNA-splicing factor ATP-dependent DLBCL RNA helicase DHX15
(DHX15) Polyadenylate-binding protein 1 (PABPC1) myeloid neoplasms
Key: MDS = Myelodysplastic syndrome AML = Acute Myeloid Leukemia
CMML = chronic myelomonocytic leukemia LUAD = Lung adenocarcinoma
UCEC = Uterine Corpus Endometrial Carcinoma PMF = Progressive
Massive Fibrosis PRAD = Prostate adenocarcinoma COAD = Colon
adenocarcinoma OV = Ovarian serous cystadenocarcinoma SKCM = Skin
Cutaneous Melanoma LUSC = Lung squamous cell carcinoma STAD =
Stomach adenocarcinoma GBM = Glioblastoma multiforme LGG = Brain
Lower Grade Glioma DLBCL = Diffuse Large B-Cell Lymphoma
[0362] In some embodiments, the subject's cancer may be responsive
to a method of treatment comprising administration of a compound
targeting this protein and/or the spliceosome even in the absence
of such mutations in a spliceosome gene or protein.
[0363] Screening or testing for the mutations may be carried out by
any known means, for example, genotyping, phenotyping, etc., by way
of nucleic acid amplification, electrophoresis, microarrays, blot,
functional assays, immunoassays, etc. Methods of screening may
include, for example, collecting a biological sample from said
subject containing the cancerous cells/tissue.
[0364] In some embodiments, a subject having cancer as described
herein can be treated with at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and at least one additional therapy.
[0365] In some embodiments, the at least one additional therapy
comprises a cytokine or cytokine analog therapy, e.g., any cytokine
or cytokine analog therapy disclosed herein. Cytokines are a broad
category of small proteins shown to be involved in autocrine
signaling, paracrine signaling, and/or endocrine signaling as
immunomodulating agents. Exemplary cytokines are disclosed herein,
and include chemokines, interferons, interleukins, lymphokines, and
tumor necrosis factors. As used herein, the term "cytokine" refers
to a polypeptide secreted from a cell that influences the function
of other cells to mediate an immune response, and the term
"cytokine therapy" refers to the administration and/or induction of
secretion of such a peptide. In some embodiments, the cytokine is a
recombinant cytokine or an analog thereof. In some embodiments, the
cytokine is a cytokine analog. The terms "cytokine analog" and
"cytokine analog therapy" refer to a modified cytokine, wherein one
or more amino acid residues of a native cytokine have been
substituted with other natural or unnatural amino acid residues
and/or wherein one or more natural or unnatural amino acid residues
have been added to a native cytokine. In some embodiments, a
cytokine or cytokine analog therapy comprises administering at
least one cytokine or cytokine analog to a patient in need of such
treatment.
[0366] In some embodiments, the at least one additional therapy
comprises one or more engineered tumor-targeting T-cells (e.g.,
CAR-T or other cell-based therapy), e.g., any CAR-T therapy
disclosed herein. The terms "CAR-T" and "CAR-T therapy" are used
interchangeably to refer to a CAR-modified cell or cell population
(e.g., a T-cell or T-cell population). In some embodiments, a
chimeric T-cell receptor (CAR) can be engineered using antigen
recognition sequences such that when the CAR is expressed on a cell
(e.g., a T-cell), the CAR and/or cell is reactive with the target
antigen. For instance, in some embodiments, a CAR may be engineered
by first identifying antibodies that recognize a cell-surface
expressed antigen protein domain. The antigen recognition sequences
of such antibodies can then be fused to a T-cell receptor domain
for selective targeting and activation. In some embodiments, the
CAR sequences are cloned into patient-derived T-cell populations
and expanded using currently available protocols. In some
embodiments, the engineered T-cells are then transfused back into
the patient's circulation, before, simultaneously with, or
following treatment with at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing. After treatment with the at least one compound and/or
pharmaceutically acceptable salt, in some embodiments, the tumor
cells may begin to present an antigen, e.g., an antigen targeted by
the engineered T-cell population. In some embodiments, the
engineered T-cell population can engage with and kill antigen
presenting tumor cells.
[0367] In some embodiments, the at least one additional therapy
comprises a checkpoint inhibitor therapy, e.g., any checkpoint
inhibitor therapy disclosed herein. Immune checkpoints are
inhibitory pathways that slow down or stop immune reactions and
prevent excessive tissue damage from uncontrolled activity of
immune cells. As used herein, the terms "checkpoint inhibitor" and
"checkpoint inhibitor therapy" are used interchangeably to refer to
any therapeutic agent, including any small molecule chemical
compound, antibody, nucleic acid molecule, or polypeptide, or any
fragments thereof, that inhibits one or more of the inhibitory
pathways, thereby allowing more extensive immune activity. In some
embodiments, a checkpoint inhibitor therapy comprises administering
at least one checkpoint inhibitor to a patient in need of such
treatment.
[0368] In some embodiments, the at least one additional therapy
comprises a neoantigen vaccine. In some embodiments, treatment
comprises administering at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing and
administering a neoantigen vaccine. In some embodiments, the
neoantigen vaccine comprises a tumor neoantigen and/or a neoantigen
induced by the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, treatment further comprises administering a checkpoint
inhibitor therapy. In some embodiments, the checkpoint inhibitor
therapy is targeted at PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3,
GITR, and/or MR. In some embodiments, the checkpoint inhibitor
therapy is targeted at PD1/PDL1 (e.g., an anti-PD1 antibody or an
anti-PDL1 antibody). In some embodiments, the checkpoint inhibitor
therapy is targeted at CTLA4 (e.g., an anti-CTLA4 antibody). In
some embodiments, treatment comprises administering a combination
therapy comprising a neoantigen vaccine after first (i)
administering at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing; and (ii)
detecting the presence of a neoantigen (e.g., a neoantigen from the
neoantigen vaccine). In some embodiments, neoantigen expression is
monitored during the course of treatment. In some embodiments,
treatment is discontinued if neoantigens are not detected.
[0369] Also disclosed herein, in some embodiments, are methods of
treating a patient by inducing neoantigens in tumor cells that can
be targeted by the patient's immune system for clearance. Without
being bound by theory, in some embodiments, administering at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, may produce neoantigens
that induce an immune response, induce a double-stranded RNA immune
response, e.g., as a result of re-expressed intron-resident
endogenous retroviruses, and/or produce neoantigens that induce
immunogenic cell death.
[0370] As used herein, the term "neoantigen" refers to any antigen
to which the immune system has not previously been exposed that
arises from one or more tumor-specific mutations and/or from
exposing a tumor to at least one compound chosen from at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing. Tumor-specific mutations can include
missense mutations, frameshifts, translocations, and mRNA splicing
variants, as well as mutations that influence posttranslational
processing, such as phosphorylation and glycosylation. These
exemplary mutations, in some embodiments, can be derived from
non-synonymous coding changes and/or mutations that alter mRNA
processing (e.g., splicing). All of these exemplary mutations, in
some embodiments, can result in molecular changes that can be
discriminated by an appropriate T-cell receptor. In some
embodiments, an exemplary neoantigen is a neoantigen induced by
delivery of at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, delivery of at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing can induce novel mRNA splicing that results in the
translation of proteins containing one or more novel peptide
domains to which the immune system has not previously been exposed.
In some embodiments, tumor-specific mutations may be mRNA splicing
variants resulting from delivery or administration of at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing.
[0371] Without being bound by theory, in some embodiments, the
delivery of at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing may
induce novel mRNA splicing (e.g., exon skipping, intron retention)
that results in the alteration of the open reading frames and/or
coding sequences of various genes. In some embodiments, these
altered genes are translated into proteins containing one or more
novel peptide domains recognized by the immune system as foreign.
In some embodiments, the one or more novel peptide domains do not
exist in the proteins or in any other part of the human proteome in
the absence of compound treatment. In some embodiments, the
proteins containing the one or more novel peptide domains can be
degraded by the proteasome to create novel peptide fragments that
act as substrates for the immunopeptide presentation machinery,
e.g., via MHC presentation. In some embodiments, the novel peptide
fragments representing neoantigens can be presented in the
MHC1-bound peptidome, e.g., on tumor cells.
[0372] In some embodiments, the delivery of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing may lead to one or more tumor cell-intrinsic
events (e.g., cell growth arrest). In some embodiments, the tumor
cell-intrinsic event(s) may lead to (1) enhanced engagement by
phagocytic cells (Bracci et al. (2014) Cell Death Differ.
21(1):15-25); (2) the transport of novel peptide fragments to a
tumor draining lymph node to engage with antigen-presenting cells;
(3) antigen-presenting cells processing novel peptide fragments
from a phagocytosed tumor cell and presenting the fragments as
neoantigens to circulating naive T-cell populations; (4) novel
peptide fragments interacting with T-cells expressing receptors
that recognize the fragments as neoantigens; (5) maturation and
activation of effector T-cell responses (e.g., CD4+ and/or CD8+
T-cells; and/or (6) engagement of T-cells with additional tumor
cells exposed to the compound treatment and presenting novel
peptide fragments representing neoantigens on their surface MHC1
complexes. In some embodiments, the tumor cell-intrinsic event(s)
may result, either directly or indirectly, in T-cell engagement of
effector function and/or killing of neoantigen-presenting tumor
cells.
[0373] Also, without being bound by theory, in some embodiments,
the delivery of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing may cause
the re-expression of intron-resident endogenous retroviruses,
leading to a double-stranded RNA immune response.
[0374] Further, without being bound by theory, in some embodiments,
the delivery of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing may lead
to immunogenic cell death triggered by compound-induced release of
mutationally-derived neoantigens. In some embodiments, the delivery
of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing may
induce a double-stranded RNA immune response. In some embodiments,
the double-stranded RNA immune response can result from the
re-expression of intron-resident endogenous retroviruses. In some
embodiments, the double-stranded RNA immune response can result in
tumor cell death. In some embodiments, the delivery of at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing may induce immunogenic cell death. In some
embodiments, the immunogenic cell death can result from release of
mutational-derived neoantigens and/or a host immune response
against tumor cells.
[0375] Accordingly, in some embodiments, methods of treatment are
disclosed comprising inducing neoantigens by administering at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing. In some embodiments, the
method comprises administering a reduced dosage of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing than would be needed absent the induction
of neoantigens. In some embodiments, the method comprises
administering one or more initial induction doses to produce
neoantigens and induce an immune response (e.g., converting naive
T-cells to memory cells), followed by a reduced dosage or
administration frequency (i.e., because of the combinatorial effect
of the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing and of
immune targeting of the neoantigens). In some embodiments,
treatment can comprise a combination of administering the at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing to induce a
neoantigen-based immune response and at least one additional
therapy (e.g., a second anti-cancer therapy). For example, in some
embodiments, treatment can comprise a combination of administering
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing to induce
a neoantigen-based immune response and one or more checkpoint
inhibitors. In some embodiments, treatment can comprise a
combination of administering the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing to induce a neoantigen-based immune response and one or
more cytokines or cytokine analogs. In some embodiments, treatment
can comprise a combination of administering the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing to induce a neoantigen-based immune
response and one or more neoantigen vaccines. In some other
embodiments, treatment can comprise a combination of administering
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing to induce
a neoantigen-based immune response and one or more engineered
tumor-targeting T-cells (e.g., CAR-T).
[0376] In some embodiments, neoantigens can be used to monitor the
effectiveness of treatment with at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing. For instance, after administration of at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, a patient sample (e.g., a tumor biopsy)
can be obtained and screened for neoantigens or for identifiers of
an immune or inflammatory response. Further treatment can be
provided, e.g., at reduced dosage, if a neoantigen and/or immune
response is detected.
[0377] In some embodiments, methods of treatment are disclosed
comprising inducing a double-stranded RNA immune response by
administering at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing.
[0378] In some embodiments, methods of treatment are disclosed
comprising inducing immunogenic cell death by administering at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing.
[0379] In some embodiments, administration of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing can be combined with any known anti-cancer
therapy. Examples of current immune activating strategies available
for oncology treatment include, but are not limited to, treatment
with immune checkpoint inhibitor (ICI) molecules, treatment with
cytokines or cytokine analogs, vaccination with tumor-associated
vaccines, and engineering tumor-targeting T-cells (e.g., expansion
of tumor-infiltrating lymphocytes or CAR-T). These technologies are
predominantly focused on enhancing or inducing an immune response
to already existing tumor antigens (either mutations or aberrant
expression of cell-surface proteins). One or more of these
strategies may involve one or more mutations that are capable of
inducing an antigenic T-cell response. For example, patient
responses to checkpoint inhibition may correlate with
non-synonymous mutational burden. In addition, cancer vaccine
approaches may be used that rely on pre-existing mutations and the
antigenicity of these mutations.
[0380] Compounds of Formula I, compounds of Formula II, compounds
of Formula III, and pharmaceutically acceptable salts of any of the
foregoing may induce broad-ranging changes in the transcriptome
that occur in multiple lineages. Translation of these mRNA changes
may produce robust and reproducible protein changes that produce
MHC1-bound neopeptides with high affinity across multiple HLA
isotypes. Without being bound by theory, due to the large number of
changes to the transcriptome and proteome, treatment with at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing may enrich the number of
potentially reactive neoantigens for enhanced engagement of the
adaptive immune response.
[0381] In some embodiments, the present disclosure provides a
method of inducing at least one neoantigen by contacting a
neoplastic cell with a therapeutically effective amount of at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing. In some embodiments, the
present disclosure provides a method of inducing a double-stranded
RNA immune response by contacting a neoplastic cell with a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing. In some embodiments, the present disclosure provides a
method of inducing immunogenic cell death by contacting a
neoplastic cell with a therapeutically effective amount of at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing.
[0382] In some embodiments, the neoplastic cell is present in an in
vitro cell culture. In some embodiments, the neoplastic cell is
obtained from a subject. In some embodiments, the neoplastic cell
is present in a subject. In some embodiments, the neoplastic cell
is derived from a hematological malignancy or a solid tumor. In
some embodiments, the hematological malignancy is chosen from a
B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In some
embodiments, the hematological malignancy is chosen from acute
myeloid leukemia and multiple myeloma. In some embodiments, the
solid tumor is chosen from breast cancer (e.g., HER2-positive
breast cancer), gastric cancer (e.g., gastric adenocarcinoma),
prostate cancer, ovarian cancer, lung cancer (e.g., lung
adenocarcinoma), uterine cancer (e.g., uterine serous endometrial
carcinoma), salivary duct carcinoma, melanoma, colon cancer, and
esophageal cancer. In some embodiments, the solid tumor is chosen
from HER2-positive breast cancer, gastric adenocarcinoma, and
prostate cancer.
[0383] In some embodiments, the present disclosure further provides
a method of inducing at least one neoantigen and/or a T-cell
response in a subject having or suspected of having a neoplastic
disorder by administering to the subject a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. Also
provided herein, in some embodiments, is a method of treating a
subject having or suspected of having a neoplastic disorder by
administering to the subject a therapeutically effective amount of
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, wherein administration of
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing induces
at least one neoantigen and/or a T-cell response.
[0384] In various other embodiments, the present disclosure
provides a method of inducing a double-stranded RNA immune response
in a subject having or suspected of having a neoplastic disorder by
administering to the subject a therapeutically effective amount of
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing. Also provided herein, in
some embodiments, is a method of treating a subject having or
suspected of having a neoplastic disorder by administering to the
subject a therapeutically effective amount of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing, wherein administration of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing induces a double-stranded RNA immune
response.
[0385] In still other embodiments, the present disclosure provides
a method of inducing immunogenic cell death in a subject having or
suspected of having a neoplastic disorder by administering to the
subject a therapeutically effective amount of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. Further provided herein, in some embodiments,
is a method of treating a subject having or suspected of having a
neoplastic disorder by administering to the subject a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing comprising at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, wherein
administration of the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing
induces immunogenic cell death.
[0386] In some embodiments, the present disclosure further provides
a method of treating a subject having or suspected of having a
neoplastic disorder by administering to the subject a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, wherein administration of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing induces immunogenic cell death, in combination
with one or more additional therapies comprising a second
agent.
[0387] In some embodiments of the therapeutic methods described
herein, the amount of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, or second agent administered is reduced due to induction
of at least one neoantigen and/or a T-cell response, as compared to
a standard dosage of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, or second agent. In some embodiments, the administered
amount of the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, or
second agent is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 75%, or 90%, as compared to a standard dosage of the at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, or second agent. In some
embodiments, the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, or
second agent is administered at least 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 75%, or 90% less frequently, as compared to a
standard dosing regimen of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, or second agent. In some embodiments, the administered
amount and/or dosage of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, or second agent results in lower systemic toxicity
and/or improved tolerance.
[0388] As used herein, the term "standard dosage" or "standard
dosing regimen" refers to any usual or routine dosing regimen for a
therapeutic agent, e.g., a regimen proposed by the manufacturer,
approved by regulatory authorities, or otherwise tested in human
subjects to meet the average patient's needs. In some embodiments,
the therapeutic agent is at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing with anti-cancer activity.
[0389] For instance, a standard dosing regimen for trastuzumab, an
exemplary anti-HER2 antibody, may be 8 mg/kg administered
intravenously over 90 min (week 1) followed by 6 mg/kg administered
intravenously over 30-90 min every 3 weeks (week 4 through the end
of the therapy cycle) (Herceptin.RTM. (trastuzumab) FDA Label
Supplement, 2017).
[0390] As another example, a standard dosing regimen for
ipilimumab, an exemplary anti-CTLA4 checkpoint inhibitor antibody,
may be 3 mg/kg administered intravenously over 90 min every 3 weeks
for 4 doses (Yervoy.RTM. (ipilimumab) FDA Label Supplement, 2018).
Another standard dosing regimen for ipilimumab may be 10 mg/kg
administered intravenously over 90 min every 3 weeks for 4 doses,
followed by 10 mg/kg every 12 weeks for up to 3 years (Yervoy.RTM.
(ipilimumab) FDA Label Supplement, 2018).
[0391] As another example, a standard dosing regimen for nivolumab,
an exemplary anti-PD1 checkpoint inhibitor antibody, may be 3 mg/kg
administered intravenously over 60 min every 2 weeks (Opdivo.RTM.
(nivolumab) FDA Label, 2015).
[0392] As another example, a standard dosing regimen for
atezolizumab, an exemplary anti-PDL1 checkpoint inhibitor antibody,
may be 1200 mg administered intravenously over 60 min every 3 weeks
(Tecentriq.RTM. (atezolizumab) FDA Label Supplement, 2018).
[0393] As yet another example, a standard dosing regimen for T-DM1,
an exemplary anti-HER2 antibody-drug conjugate, may be 3.6 mg/kg
administered intravenously over 90 min every 3 weeks (Kadcyla.RTM.
(T-DM1) FDA Label Supplement, 2016).
[0394] In some embodiments, the methods described herein may
further comprise administering at least one additional therapy
(e.g., a checkpoint inhibitor, a neoantigen vaccine, a cytokine or
cytokine analog, CAR-T, etc.). In some embodiments, the amount of
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and/or
the at least one additional therapy administered is reduced due to
induction of at least one neoantigen and/or a T-cell response, as
compared to a standard dosage of the at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and/or the at least one additional therapy. In some
embodiments, the amount of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and/or the at least one additional therapy administered
is reduced due to induction of a double-stranded RNA immune
response, as compared to a standard dosage of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, and/or the at least one additional
therapy. In some embodiments, the amount of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, and/or the at least one additional therapy
administered is reduced due to induction of immunogenic cell death,
as compared to a standard dosage of the at least one compound
chosen from at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and/or
the at least one additional therapy. In some embodiments, the
administered amount of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and/or the at least one additional therapy is reduced by
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as
compared to a standard dosage of the at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and/or the at least one additional therapy. In some
embodiments, the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and/or
the at least one additional therapy is administered at least 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90% less
frequently, as compared to a standard dosing regimen of the at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, and/or the at least one
additional therapy. In some embodiments, the administered amount
and/or dosage of the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and/or
the at least one additional therapy results in lower systemic
toxicity and/or improved tolerance.
[0395] In some embodiments, administration of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing is initiated before administration of the
at least one additional therapy. In other embodiments,
administration of the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing is
initiated after administration of the at least one additional
therapy. In still other embodiments, administration of the at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing is initiated concurrently
with administration of the at least one additional therapy.
[0396] In some embodiments, administration of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing is repeated at least once after initial
administration. In some embodiments, the amount of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing used for repeated administration is reduced
as compared to the amount used for initial administration. In some
embodiments, the amount of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing used for repeated administration is reduced as compared
to a standard dosage of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing. In some embodiments, the amount of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing used for repeated administration is reduced
by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, as
compared to a standard dosage or initial dosage of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing.
[0397] In some embodiments, administration of the at least one
additional therapy is repeated at least once after initial
administration. In some embodiments, the amount of the at least one
additional therapy used for repeated administration is reduced as
compared to the amount used for initial administration. In some
embodiments, the amount of the at least one additional therapy used
for repeated administration is reduced as compared to a standard
dosage of the at least one additional therapy. In some embodiments,
the amount of the at least one additional therapy used for repeated
administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 75%, or 90%, as compared to a standard dosage or initial
dosage of the at least one additional therapy.
[0398] In some embodiments, repeated administration of the at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing is concurrent with
repeated administration of the at least one additional therapy. In
some embodiments, administration of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing is sequential or staggered with repeated
administration of the at least one additional therapy.
[0399] In some embodiments, the at least one additional therapy
comprises administering a checkpoint inhibitor, e.g., any
checkpoint inhibitor disclosed herein. In some embodiments, the
subject is intolerant, non-responsive, or poorly responsive to the
checkpoint inhibitor when administered alone. In some embodiments,
the checkpoint inhibitor is targeted at PD1/PDL1, CTLA4, OX40,
CD40, LAG3, TIM3, GITR, and/or MR. In some embodiments, the
checkpoint inhibitor is targeted at CTLA4, OX40, CD40, and/or GITR.
In some embodiments, the checkpoint inhibitor is an antibody having
inhibitory or agonist activity to its target. In some embodiments,
a checkpoint inhibitor is targeted with an inhibitory antibody or
other similar inhibitory molecule. In other embodiments, a
checkpoint inhibitor is targeted with an agonist antibody or other
similar agonist molecule.
[0400] In some other embodiments, the at least one additional
therapy comprises administering a neoantigen vaccine, e.g., any
neoantigen vaccine disclosed herein. In some embodiments, the at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing is administered before
administration of the neoantigen vaccine. In some embodiments, the
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing is administered after
administration of the neoantigen vaccine. In some embodiments, the
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing is administered
concurrently with administration of the neoantigen vaccine. In some
embodiments, administration of the at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing is repeated at least once after initial administration.
In some embodiments, the amount of the at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing used for repeated administration is reduced as compared
to the amount used for initial administration.
[0401] In some embodiments, the neoantigen vaccine comprises at
least one neoantigen peptide. In some embodiments, the at least one
neoantigen peptide ranges from about 10 to about 50 amino acids in
length. In some embodiments, the at least one neoantigen peptide
ranges from about 10 to about 35 amino acids in length. In some
embodiments, the at least one neoantigen peptide ranges from about
15 to about 25 amino acids in length. In some embodiments, the at
least one neoantigen peptide comprises one or more than one
neoantigen sequence.
[0402] In some embodiments, the neoantigen sequence and/or
antigenic portion ranges from about 10 to about 50 amino acids in
length. In some embodiments, the at least one neoantigen peptide
ranges from about 10 to about 35 amino acids in length. In some
embodiments, the neoantigen sequence and/or antigenic portion
ranges from about 15 to about 25 amino acids in length. In some
embodiments, the neoantigen sequence and/or antigenic portion
ranges from about 10 to about 20 amino acids in length. In some
embodiments, the neoantigen sequence and/or antigenic portion does
not exclusively overlap or consist of the canonical peptide
sequence (e.g., any of the exemplary canonical peptide sequences
underlined in Table 13).
[0403] The term "antigenic portion" or "antigenic fragment" of a
neoantigen sequence, as used herein, refers to one or more
fragments of a neoantigen sequence that retain the ability to
induce a T-cell response (e.g., antigen-specific expansion and/or
maturation of effector T-cell population(s)). An antigenic portion,
in some embodiments, may also retain the ability to be
internalized, processed, and/or presented by antigen-presenting
cells (e.g., dendritic cells). In some embodiments, an antigenic
portion also retains T-cell priming function. In some embodiments,
an antigenic portion of a neoantigen sequence ranges from about 10
to about 50 amino acids in length. In some embodiments, an
antigenic portion of a neoantigen sequence ranges from about 10 to
about 35 amino acids in length. In some embodiments, an antigenic
portion of a neoantigen sequence ranges from about 15 to about 25
amino acids in length. In some embodiments, an antigenic portion of
a neoantigen sequence ranges from about 10 to about 20 amino acids
in length. In some embodiments, an antigenic portion of a
neoantigen sequence (e.g., an antigenic portion of any one of SEQ
ID NOs: 30-57), or its encoding mRNA, is formulated as a neoantigen
vaccine.
[0404] An exemplary embodiment of an antigenic portion is the
region(s) flanking amino acids 45-53 of SEQ ID NO: 30. Another
exemplary embodiment of an antigenic portion is the region(s)
flanking amino acids 82-90 of SEQ ID NO: 30. In some embodiments,
the antigenic portion is capable of binding to at least one HLA
allele expressed in a subject (e.g., HLA-A*02:01). In some other
embodiments, the antigenic portion is capable of binding to at
least one HLA allele expressed in at least 10%, at least 15%, at
least 20%, at least 25%, at least 30%, at least 35%, at least 40%,
or at least 45% of subjects in a population of subjects suffering
from a neoplastic disorder. In some embodiments, the antigenic
portion is capable of eliciting a T-cell response against a tumor
present in at least 1%, at least 5%, or at least 10% of a
population of subjects suffering from a neoplastic disorder.
[0405] In some embodiments, an antigenic portion does not
exclusively overlap or consist of a canonical peptide sequence. The
term "canonical peptide sequence," as used herein, refers to any
contiguous peptide sequence present in the human proteome in the
absence of contact with at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing
(e.g., in the absence of contact with at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing), and/or to which the immune has previously been exposed.
In some embodiments, the canonical peptide sequence is derived from
and/or encoded by the canonical transcript open reading frame.
Exemplary canonical peptide sequences are underlined in Table
13.
[0406] In some embodiments, when at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing is administered, a canonical peptide sequence may be
derived from and/or encoded by the immediate 5' in-frame 24
nucleotides preceding an aberrant splicing event induced by the at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing. Thus, in some
embodiments, the canonical peptide sequence comprises or consists
of the 8 amino acids immediately N-terminal to the neoantigen
sequence induced by the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing. In
some embodiments, when a 5' exon sequence terminates with a
terminal nucleotide of a codon, the canonical peptide sequence
terminates at the end of the exon. In some other embodiments, when
a 5' exon sequence terminates with one or two of the three
nucleotides of a codon, the canonical peptide sequence is derived
from and/or encoded by the 24 nucleotides preceding the incomplete
codon. In some embodiments, mRNA sequences 3' of the aberrant
splicing event may be translated in the same open reading frame
derived from the 5' exon until reaching a stop codon, whereupon
translation may terminate. In some embodiments, when the aberrant
splicing event (e.g., exon skipping) results in a conservation of
the canonical transcript open reading frame, the C-terminal
sequence may be translated for an additional 24 nucleotides,
encoding 8 C-terminal amino acids. In this context, in some
embodiments, only the region across the aberrant exon junction may
encode a neoantigen sequence. In some embodiments, when the open
reading frame is shifted (e.g., intron retention), the complete
C-terminal sequence (encoded by the 3' mRNA) may encode a
neoantigen sequence.
[0407] In some embodiments, an antigenic portion of a neoantigen
sequence is chosen by comparing the neoantigen sequence to the
canonical peptide sequence; and selecting a portion of the
neoantigen sequence that does not exclusively overlap, consist of,
and/or align with the canonical peptide sequence. An antigenic
portion of a neoantigen sequence, in some embodiments, can be
screened for antigenicity and/or T-cell priming function in the
same manner as are full-length neoantigen sequences (e.g., the
neoantigen sequence from which the antigenic portion is derived).
In some embodiments, an antigenic portion of a neoantigen sequence
is evaluated for antigenicity and/or T-cell priming function using
a T-cell priming assay, such as the exemplary T-cell priming
experiments described herein.
[0408] In some embodiments, the neoantigen sequence is a neoantigen
sequence specific to the subject. In some embodiments, the
neoantigen sequence is a personalized neoantigen vaccine for the
subject. In some embodiments, the neoantigen sequence used to
create a personalized neoantigen vaccine for a subject is capable
of binding to at least one HLA allele expressed in the subject. In
some embodiments, a personalized neoantigen vaccine is selected by
identifying neoantigens expressed in a subject's tumor, e.g., after
administration of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and
selecting a vaccine comprising a neoantigen sequence observed in
the patient's tumor.
[0409] The term "personalized" when used to describe a neoantigen
vaccine refers to a vaccine created by identifying one or more
neoantigens produced in a patient, preferably one identified in the
patient after an exposure to at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and then using one or more of those neoantigens as the
basis of the vaccine for the same patient. Accordingly, in some
embodiments, a patient is given at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing and screened for neoantigens produced by the treatment.
In some embodiments, the selected neoantigen vaccine comprises a
neoantigen peptide or mRNA disclosed herein and confirmed to be
present in the patient after exposure to the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. In some embodiments, the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing and/or peptide or mRNA vaccine may be
administered to the patient once or repeatedly. Subsequently, in
some embodiments, one or more of those neoantigens are used to
create a personalized vaccine that is given to the patient. In some
embodiments, the one or more neoantigens used to create a
personalized vaccine possess binding affinity for one or more
patient-specific HLA alleles. In some embodiments, the patient
expresses one or more MHC1 alleles that bind to the one or more
neoantigens. The prediction of whether a given neoantigen will bind
to a specific MHC1 allele can be determined using any computational
prediction method known in the art. Exemplary computational
prediction methods are disclosed, e.g., in Meydan et al. (2013) BMC
Bioinformatics 14 (Suppl. 2):513, which is incorporated herein by
reference for such methods.
[0410] In some other embodiments, the neoantigen sequence is a
universal neoantigen sequence. In some embodiments, the neoantigen
sequence is a universal neoantigen vaccine.
[0411] The term "universal" when used to describe a neoantigen
vaccine refers to a vaccine having a peptide or mRNA sequence that
is based on common or known neoantigen(s) observed by sequencing
neoantigens produced in multiple patients and/or patient tissue
samples, preferably after an exposure to at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. The peptide or mRNA sequence used in the
vaccine need not be present in every patient but rather be observed
in at least several patients or patient tissue samples. In some
embodiments, the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing and/or
peptide or mRNA vaccine may be administered to the patient once or
repeatedly. Subsequently, in some embodiments, that peptide or mRNA
sequence is used for vaccinating further patients. In some
embodiments, a patient is given at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and then given a peptide or mRNA vaccine of known
neoantigen to enhance immune response to the neoantigens produced
by the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, a patient is given a universal peptide or mRNA vaccine
and then given at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, the neoantigen sequence (or sequences) used to create
a universal neoantigen vaccine is selected based on overall MHC1
allele frequency in a given patient population (Maiers et al.
(2007) Hum. Immunol. 68(9):779-88).
[0412] In some embodiments, the neoantigen (e.g., a universal
neoantigen) sequence is capable of binding to at least one HLA
allele expressed in at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% of subjects in a population of subjects suffering from the
neoplastic disorder. In some embodiments, the neoantigen sequence
is capable of eliciting a T-cell response against a tumor present
in at least 1%, at least 5%, or at least 10% of a population of
subjects suffering from the neoplastic disorder.
[0413] In some embodiments, the neoantigen sequence has been
identified by sequencing at least one neoantigen peptide, or its
encoding mRNA, induced in the subject by administering a
therapeutically effective amount of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. In some embodiments, the at least one
neoantigen peptide comprises a neoantigen sequence induced by
contacting a neoplastic cell with a therapeutically effective
amount of the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, the neoplastic cell is present in an in vitro cell
culture. In some embodiments, the neoplastic cell is obtained from
the subject. In some embodiments, the neoplastic cell is present in
the subject.
[0414] In some embodiments, the neoantigen vaccine comprises at
least one neoantigen peptide and a pharmaceutically acceptable
carrier (e.g., any of the exemplary carriers described herein). In
some embodiments, the at least one neoantigen peptide is linked to
the pharmaceutically acceptable carrier. In some embodiments, the
pharmaceutically acceptable carrier is chosen from a peptide, a
serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a
thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid
derivative, a cytokine, and a chemokine. In some embodiments, the
neoantigen peptide and the pharmaceutically acceptable carrier are
covalently attached via a linker. In some embodiments, the
neoantigen peptide and the pharmaceutically acceptable carrier are
expressed as a fusion protein. In some embodiments, the neoantigen
vaccine comprises at least one neoantigen peptide and a
pharmaceutically acceptable diluent. In some embodiments, the
neoantigen vaccine comprises at least one neoantigen peptide and a
pharmaceutically acceptable adjuvant.
[0415] In some embodiments, the neoantigen vaccine comprises at
least one neoantigen mRNA. In some embodiments, the at least one
neoantigen mRNA encodes one or more than one neoantigen
sequence.
[0416] In some embodiments, the neoantigen sequence is a neoantigen
sequence specific to the subject. In some embodiments, the
neoantigen sequence is a personalized neoantigen vaccine for the
subject. In some embodiments, the neoantigen sequence is capable of
binding to at least one HLA allele expressed in the subject.
[0417] In some other embodiments, the neoantigen sequence is a
universal neoantigen sequence. In some embodiments, the neoantigen
sequence is a universal neoantigen vaccine. In some embodiments,
the neoantigen sequence is capable of binding to at least one HLA
allele expressed in at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% of subjects in a population of subjects suffering from the
neoplastic disorder. In some embodiments, the neoantigen sequence
is capable of eliciting a T-cell response against a tumor present
in at least 1%, at least 5%, or at least 10% of a population of
subjects suffering from the neoplastic disorder.
[0418] In some embodiments, the neoantigen sequence has been
identified by sequencing the protein sequence of at least one
neoantigen. In some embodiments, the neoantigen sequence has been
identified by sequencing at least one mRNA encoding a neoantigen
induced in the subject by administering a therapeutically effective
amount of the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, the at least one neoantigen mRNA encodes a neoantigen
sequence induced by contacting a neoplastic cell with a
therapeutically effective amount of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. In some embodiments, the neoplastic cell is
present in an in vitro cell culture. In some embodiments, the
neoplastic cell is obtained from the subject. In some embodiments,
the neoplastic cell is present in the subject.
[0419] In some embodiments, the neoantigen vaccine comprises at
least one neoantigen mRNA and a pharmaceutically acceptable carrier
(e.g., any of the exemplary carriers described herein). In some
embodiments, the at least one neoantigen mRNA is linked to the
pharmaceutically acceptable carrier. In some embodiments, the
pharmaceutically acceptable carrier is chosen from a peptide, a
serum albumin, a keyhole limpet hemocyanin, an immunoglobulin, a
thyroglobulin, an ovalbumin, a toxoid or an attenuated toxoid
derivative, a cytokine, and a chemokine. In some embodiments, the
neoantigen vaccine comprises at least one neoantigen mRNA and a
pharmaceutically acceptable diluent. In some embodiments, the
neoantigen vaccine comprises at least one neoantigen mRNA and a
pharmaceutically acceptable adjuvant. In some embodiments, the
neoantigen mRNA is encapsulated by an encapsulating agent. In some
embodiments, the encapsulating agent is a liposome. In some
embodiments, the encapsulating agent is a nanoparticle.
[0420] In some embodiments, the at least one additional therapy
comprises administering a cytokine or cytokine analog, e.g., any
cytokine or cytokine analog disclosed herein. In some embodiments,
the subject is intolerant, non-responsive, or poorly responsive to
the cytokine or cytokine analog when administered alone. In some
embodiments, the cytokine or cytokine analog comprises a T-cell
enhancer. In some embodiments, the cytokine or cytokine analog
comprises IL-2, IL-10, IL-12, IL-15, IFN.gamma., and/or TNF.alpha..
In some embodiments, the cytokine or cytokine analog comprises
IL-2, IL-10, IL-12, and/or IL-15. In some embodiments,
administering the cytokine or cytokine analog enhances T-cell
priming following administration of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing due to the induction and presentation of neoantigens.
[0421] In some embodiments, the at least one additional therapy
comprises administering engineered tumor-targeting T-cells (i.e.,
CAR-T), e.g., any CAR-T therapy disclosed herein.
[0422] In some embodiments, the methods described herein may
further comprise detecting one or more neoantigens and/or a T-cell
response in the subject after administration of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, and, optionally, continuing administration
of the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing if one or
more neoantigens and/or a T-cell response is detected. In some
embodiments, detecting one or more neoantigens and/or a T-cell
response in the subject indicates efficacy of treatment with the at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing. In some embodiments,
treatment with the additional therapy, along with the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, is continued if one or more neoantigens
and/or a T-cell response is detected. In some embodiments,
treatment is continued at a reduced dosage and/or frequency if one
or more neoantigens and/or a T-cell response is detected.
[0423] In some embodiments, the methods described herein may
further comprise detecting a double-stranded RNA immune response in
the subject after administration of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing, and, optionally, continuing administration of
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing if a
double-stranded RNA immune response is detected. In some
embodiments, detecting a double-stranded RNA immune response in the
subject indicates efficacy of treatment with the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing. In some embodiments, treatment with the
additional therapy, along with the at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, is continued if a double-stranded RNA immune response is
detected. In some embodiments, treatment is continued at a reduced
dosage and/or frequency if a double-stranded RNA immune response is
detected.
[0424] In some embodiments, the methods described herein may
further comprise detecting immunogenic cell death in the subject
after administration of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and, optionally, continuing administration of the at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing if immunogenic cell death
is detected. In some embodiments, detecting immunogenic cell death
in the subject indicates efficacy of treatment with the at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing. In some embodiments,
treatment with the additional therapy, along with the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, is continued if immunogenic cell death is
detected. In some embodiments, treatment is continued at a reduced
dosage and/or frequency if immunogenic cell death is detected.
[0425] In some embodiments, the subject has a non-synonymous
mutational burden of about 150 mutations or less. In some
embodiments, the subject has a non-synonymous mutational burden of
about 100 mutations or less. In some embodiments, the subject has a
non-synonymous mutational burden of about 50 mutations or less. In
some embodiments, the subject has or is suspected of having a
neoplastic disorder, e.g., a hematological malignancy or a solid
tumor. In some embodiments, the hematological malignancy is chosen
from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In
some embodiments, the hematological malignancy is chosen from acute
myeloid leukemia and multiple myeloma. In some embodiments, the
solid tumor is chosen from breast cancer, gastric cancer, prostate
cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct
carcinoma, melanoma, colon cancer, and esophageal cancer. In some
embodiments, the solid tumor is chosen from HER2-positive breast
cancer, gastric adenocarcinoma, and prostate cancer.
[0426] In some embodiments, the present disclosure further provides
a method of treating a subject having or suspected of having a
neoplastic disorder, comprising: (a) administering to the subject a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, wherein administration of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing induces at least one neoantigen and/or a
T-cell response; (b) detecting one or more neoantigens and/or a
T-cell response in the subject after administration of the at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing; and (c) continuing
administration of the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing if
one or more neoantigens and/or a T-cell response is detected. In
some embodiments, detecting one or more neoantigens and/or a T-cell
response in the subject indicates efficacy of treatment with the at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing. In some embodiments, the
one or more neoantigens comprise an amino acid sequence of any one
of SEQ ID NOs: 1-29. In some embodiments, the one or more
neoantigens comprise an amino acid sequence of SEQ ID NO: 1. In
some embodiments, the one or more neoantigens comprise an amino
acid sequence of SEQ ID NO: 3. In some embodiments, the one or more
neoantigens comprise an amino acid sequence of any one of SEQ ID
NOs: 10-13.
[0427] In some embodiments, a patient having a cancer as described
herein can be treated with a combination of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing and a checkpoint inhibitor therapy.
[0428] Treatment of patients with immune checkpoint inhibition has
been shown to have robust efficacy in certain clinical indications.
Recently, the FDA approved use of a checkpoint inhibitor in
patients with tumors exhibiting high microsatellite instability,
agnostic to the tissue lineage. This approval was based, in part,
on the observation that response rates correlate positively with
mutational burden (Rizvi et al. (2015) Science 348(6230):124-8;
Hellmann et al. (2018) Cancer Cell 33(5):853-861). Estimates from
the literature vary in absolute numbers and by lineage, but
generally support that above a threshold of .about.150-250
mutations, the probability of response rises. Analysis of TCGA data
shows that a large percentage of adult-onset tumor lineages have
comparatively low non-synonymous mutational burden (Vogelstein et
al. (2013) Science 339:1549-58). Most lineages have median
non-synonymous mutational rates of .about.30-80 per patient, well
below the thresholds for improved odds of response to checkpoint
inhibitors.
[0429] For instance, HER2-positive breast cancer has been shown to
have a median of .about.60 non-synonymous mutations present per
patient sample. However, the threshold for checkpoint inhibitor
treatment efficacy, as mentioned above, is estimated to be in the
range of .about.150-250 non-synonymous mutations, i.e., patients
above this threshold are more likely to show complete remission,
partial remission, and/or stable disease, whereas patients below
this threshold are more likely to exhibit progressive disease.
Strategies to enhance the apparent number of non-synonymous
mutations and/or neoantigens being presented on tumor cells are
therefore desirable, and may enhance the overall probability of
responses, e.g., to checkpoint inhibitor therapies. As cytokines
(and analogs thereof) act via a similar mechanism of action, such
strategies may also enhance the overall probability of response to
cytokine-based therapies.
[0430] Current response rates in HER2-positive breast cancer are
.about.15-25% (CTI NCT02129556). In some embodiments disclosed
herein, treatment with at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing in
combination with a checkpoint inhibitor and/or cytokine therapy may
improve such response rates. In some embodiments, treatment with a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, in combination with a checkpoint inhibitor and/or
cytokine therapy may apply to any adult-onset tumor, particularly
those in which the median non-synonymous mutational rate is below
the estimated .about.150 mutations threshold. In some embodiments,
exemplary cancer types suitable for treatment with a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, alone or in combination with an additional therapy
(e.g., a checkpoint inhibitor therapy, a cytokine therapy) include
but are not limited to esophageal cancer, non-Hodgkin's lymphoma,
colorectal cancer, head and neck cancer, gastric cancer,
endometrial cancer, pancreatic adenocarcinoma, ovarian cancer,
prostate cancer, hepatocellular cancer, glioblastoma, breast cancer
(e.g., HER2-positive breast cancer), lung cancer (e.g., non-small
cell lung cancer), chronic lymphocytic leukemia, and acute myeloid
leukemia. Other exemplary suitable cancer types are identified,
e.g., in Vogelstein et al. (2013) Science 339:1549-58, which is
incorporated herein by reference in its entirety.
[0431] As many checkpoint inhibitor therapies are based on chronic
expression of tumor-associated antigens, regular treatment boosts
are required for efficacy and for "re-boosting" reactive T-cell
populations. The inducible nature of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, derived neoantigens described herein provide for
therapeutic dosing regimens that may be designed to enhance the
immune response of neoantigen-reactive T-cells, while limiting
T-cell exhaustion often caused by chronic antigen stimulation. For
instance, in some embodiments, an initial dose of at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, is administered to a subject to trigger
aberrant splicing and production of neoantigen peptides. After a
period of time to allow for protein production and antigen
presentation, in some embodiments, the subject is then administered
an initial dose of a checkpoint inhibitor to boost and/or enhance
effector T-cell priming and expansion. In some embodiments, the
wait period between doses of at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and checkpoint inhibitor is about 2, about 3, about 4,
about 5, about 6, or about 7 days. In some embodiments, the wait
period is between about 3 days and about 5 days.
[0432] In some embodiments, the checkpoint inhibitor is targeted at
CTLA4, OX40, CD40, and/or GITR. In some embodiments, the
combination therapeutic benefit of a therapeutically effective
amount of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and a
checkpoint inhibitor may be additive or superadditive.
[0433] In some embodiments, administration of the therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing is
initiated before administration of the checkpoint inhibitor.
[0434] In some embodiments, administration of the therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing thereof
is initiated after administration of the checkpoint inhibitor.
[0435] In some embodiments, administration of the therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing is
initiated concurrently with administration of the checkpoint
inhibitor, e.g., in a single formulated product or separate
formulated products administered in a single procedure.
[0436] In some embodiments, after a period to allow for T-cell
priming and expansion, the subject is then administered a second or
subsequent dose of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, to
trigger re-presentation of neoantigen peptides. In some
embodiments, the wait period between an initial dose of a
checkpoint inhibitor and a second or subsequent dose of a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, is about 2, about 3, about 4, or about 5 weeks. In some
embodiments, the wait period is about 3 weeks. Following a second
or subsequent dose of at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing, in
some embodiments, the immune system may engage with the
neoantigen-presenting tumor cells and/or elicit tumor cell killing.
In some embodiments, the subject is then administered a second or
subsequent dose of the checkpoint inhibitor to further expand the
memory effector T-cell population, after allowing for secondary
T-cell priming and expansion.
[0437] In some embodiments, the wait period between an initial dose
of a therapeutically effective amount of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing and a second or subsequent dose of a
checkpoint inhibitor is about 2, about 3, about 4, or about 5
weeks. In some embodiments, the wait period is about 3 weeks.
[0438] In some embodiments, dosing of a therapeutically effective
amount of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing,
following this exemplary initial treatment regimen can be
pulsatile, i.e., a therapeutically effective amount of at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, may be dosed at prolonged intervals (e.g.,
about every 4 weeks, about every 5 weeks, about every 6 weeks) to
allow for antigen presentation, T-cell engagement and/or tumor cell
killing, and/or recovery of the memory T-cell population. At later
timepoints, in some embodiments, a therapeutically effective amount
of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing,
treatment may be combined with one or more checkpoint inhibitors
targeted to restore effector functionality to exhausted T-cell
populations. For example, in some embodiments, at later timepoints,
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing,
treatment may be combined with one or more checkpoint inhibitors
targeted at PD1/PDL1, LAG3, and/or TIM3. In some embodiments, the
pulsed nature of neoantigen presentation and priming may allow a
checkpoint inhibitor and/or at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, to be administered less frequently and/or at lower
doses. In some embodiments, the pulsed nature of neoantigen
presentation may provide one or more treatment benefits for a
checkpoint inhibitor (e.g., an anti-CTLA4 antibody such as
ipilimumab), relative to the checkpoint inhibitor when administered
without concurrent administration of a therapeutically effective
amount of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, for
example, by lowering the potential risk of adverse reactions often
observed with the checkpoint inhibitor's standard dosing regimen.
In certain embodiments, the checkpoint inhibitor is an inhibitor of
the cytotoxic T-lymphocyte-associated antigen (CTLA4) pathway.
CTLA4, also known as CD152, is a protein receptor that
downregulates immune responses. CTLA4 is constitutively expressed
in regulatory T-cells, but only upregulated in conventional T-cells
after activation. As used herein, the term "CTLA4 inhibitor" is
meant to refer to any inhibitor of CTLA4 and/or the CTLA4 pathway.
Exemplary CTLA4 inhibitors include but are not limited to
anti-CTLA4 antibodies. CTLA4 blocking antibodies for use in humans
were developed based on the pre-clinical activity seen in mouse
models of anti-tumor immunity. Exemplary anti-CTLA4 antibodies
include but are not limited to ipilimumab (MDX-010) and
tremelimumab (CP-675,206), both of which are fully human.
Ipilimumab is an IgG1 with a plasma half-life of approximately
12-14 days; tremelimumab is an IgG2 with a plasma half-life of
approximately 22 days. See, e.g., Phan et al. (2003) Proc Natl Acad
Sci USA. 100:8372-7; Ribas et al. (2005) J Clin Oncol. 23:8968-77;
Weber et al. (2008) J Clin Oncol. 26:5950-6. In some embodiments,
the anti-CTLA4 antibody is ipilimumab.
[0439] In certain embodiments, the checkpoint inhibitor is an
inhibitor of the programmed death-1 (PD1) pathway. The programmed
cell death 1 (PD1) pathway represents a major immune control switch
which may be engaged by tumor cells to overcome active T-cell
immune surveillance. The ligands for PD1 (PDL1 and PDL2) are
constitutively expressed or can be induced in various tumors. High
expression of PDL1 on tumor cells (and to a lesser extent of PDL2)
has been found to correlate with poor prognosis and survival in
various other solid tumor types. Furthermore, PD1 has been
suggested to regulate tumor-specific T-cell expansion in patients
with malignant melanoma. These observations suggest that the
PD1/PDL1 pathway plays a critical role in the tumor immune evasion
and may be considered an attractive target for therapeutic
intervention. As used herein, the term "PD1 inhibitor" is meant to
refer to any inhibitor of PD1 and/or the PD1 pathway. Exemplary PD1
inhibitors include but are not limited to anti-PD1 and anti-PDL1
antibodies. In certain embodiments, the checkpoint inhibitor is an
anti-PD1 antibody. Exemplary anti-PD1 antibodies include but are
not limited to nivolumab and pembrolizumab (MK-3475). Nivolumab,
for example, is a fully human immunoglobulin G4 (IgG4) PD1 immune
checkpoint inhibitor antibody that disrupts the interaction of the
PD1 receptor with its ligands PDL1 and PDL2, thereby inhibiting the
cellular immune response (Guo et al. (2017) J Cancer 8(3):410-6).
In some embodiments, the anti-PD1 antibody is nivolumab.
Pembrolizumab, for example, is a potent and highly-selective
humanized mAb of the IgG4/kappa isotype designed to directly block
the interaction between PD1 and its ligands, PDL1 and PDL2.
Pembrolizumab strongly enhances T lymphocyte immune responses in
cultured blood cells from healthy human donors, cancer patients,
and primates. Pembrolizumab has also been reported to modulate the
level of interleukin-2 (IL-2), tumor necrosis factor alpha
(TNF.alpha.), interferon gamma (IFN.gamma.), and other cytokines.
Exemplary anti-PDL1 antibodies include but are not limited to
atezolizumab, avelumab, and durvalumab. Atezolizumab, for example,
is an IgG1 humanized mAb that is reported to block the PD1/PDL1
interaction, by targeting the expressed PDL1 on numerous kinds of
malignant cells. This blockage of the PD1/PDL1 pathway may
stimulate the immune defense mechanisms against tumors (Abdin et
al. (2018) Cancers (Basel) 10(2):32). In some embodiments, the
anti-PDL1 antibody is atezolizumab.
[0440] In certain embodiments, the checkpoint inhibitor is targeted
at PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or MR. In
certain embodiments, the checkpoint inhibitor is targeted at CTLA4,
OX40, CD40, and/or GITR. In certain embodiments, a checkpoint
inhibitor is targeted with an inhibitory antibody or other similar
inhibitory molecule (e.g., an inhibitory anti-CTLA4 or
anti-PD1/PDL1 antibody). In certain other embodiments, a checkpoint
inhibitor is targeted with an agonist for the target; examples of
this class include the stimulatory targets OX40, CD40, and/or GITR.
In some embodiments, the checkpoint inhibitor targeted at OX40,
CD40, and/or GITR is an agonist antibody. Agonist antibodies
directed against OX40 may have a dual role, inhibiting regulatory
T-cell suppression, while enhancing effector T-cell functions.
Agonist anti-GITR antibodies have also been shown to make effector
T-cells more resistant to the inhibition induced by regulatory
T-cells (Karaki et al. (2016) Vaccines (Basel) 4(4):37). Likewise,
agonist CD40 antibodies demonstrate T-cell-dependent anti-tumor
activity. Activation of CD40 on dendritic cells increases
cross-presentation of tumor antigens and consequently the number of
activated tumor-directed effector T-cells (Ellmark et al. (2015)
Oncoimmunol. 4(7):e1011484).
[0441] In certain embodiments, the checkpoint inhibitor is targeted
at CTLA4 (e.g., an anti-CTLA4 antibody). In certain embodiments,
targeting CTLA4 facilitates priming and activation of naive
T-cells. In certain embodiments, the checkpoint inhibitor is
targeted at OX40 (e.g., an anti-OX40 antibody). In certain
embodiments, targeting OX40 enhances expansion of effector T-cells.
In certain embodiments, the checkpoint inhibitor is targeted at
CD40 (e.g., an anti-CD40 antibody). In certain embodiments,
targeting CD40 inhibits "tolerogenic" priming of T-cells and/or
formation of regulatory T-cells. In certain embodiments, the
checkpoint inhibitor is targeted at GITR (e.g., an anti-GITR
antibody). In certain embodiments, targeting GITR inhibits activity
of regulatory T-cells. In certain embodiments, the benefit of
combination therapy (e.g., the effect on at least one symptom or
the risk/rate of disease progression) with a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and a
CTLA4-, OX40-, CD40-, and/or GITR-targeted agent is additive. In
some embodiments, the benefit of combination therapy with a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and a CTLA4-, OX40-, CD40-, and/or GITR-targeted agent
is superadditive (i.e., synergistic).
[0442] Checkpoint inhibitor treatment strategies are based on the
hypothesis that treatment facilitates and/or enhances priming of
T-cell responses to weakly or poorly antigenic tumors (e.g., CTLA4)
or that treatment restores and/or reinvigorates T-cells that
respond to tumor antigens, but have become "exhausted" due to the
chronic nature of the antigen presentation (e.g., PD1, PDL1) (Chen
and Mellman (2013) Immunity 39(1):1-10). Examples of suitable
checkpoint inhibition therapies and agents, e.g., anti-PD1,
anti-PDL1, or anti-CTLA4 antibodies, are known in the art. See,
e.g., WO 2001/014424 WO 2013/173223, WO 2016/007235.
[0443] Combining these primed T-cell responses following checkpoint
inhibitor therapy with treatment to induce neoantigens in tumor
cells (e.g., by administration of a therapeutically effective
amount of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing) to which
the primer immune system can react may provide beneficial synergy.
As compounds chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing derived neoantigens have
not yet been presented for T-cell priming, combination with a CTLA4
inhibitor may be particularly beneficial. In some embodiments,
treatment comprises administering a therapeutically effective
amount of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, to
induce the production of neoantigens, followed before,
concurrently, or thereafter by an initial administration of a CTLA4
inhibitor to stimulate CD8 T-cell priming. In some embodiments,
additional administrations of a CTLA4 inhibitor are provided to the
patient, e.g., to further stimulate priming and/or activation of
neoantigen-reactive CD8 populations. In some embodiments,
additional administrations of a therapeutically effective amount of
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, can be given to the
patient to increase neoantigen presentation by the tumor. Repeat
administrations of a therapeutically effective amount of at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, and checkpoint inhibitor
therapy can occur concurrently or in staggered intervals. In some
embodiments, treatment further comprises administering a PD1/PDL1
inhibitor co-treatment, e.g., to restore effector function of
exhausted neoantigen-targeted T-cells within the tumor
microenvironment.
[0444] The terms "combination" or "combination therapy," as used
herein, refer to the administration of a therapeutically effective
amount of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, together
with an additional agent or therapy (e.g., a checkpoint inhibitor,
a cytokine or cytokine analog, a neoantigen vaccine, CAR-T), as
part of a treatment regimen intended to provide a beneficial (i.e.,
additive or synergistic) effect from the co-action of one or more
of the administered agents. In some embodiments, the combination
may also include one or more additional agents, including but not
limited to chemotherapeutic agents, anti-angiogenesis agents, and
agents that reduce immune-suppression (e.g., a second checkpoint
inhibitor). The beneficial effect of the combination includes, but
is not limited to, pharmacokinetic or pharmacodynamic co-action
resulting from the combination of therapeutic agents.
Administration of these therapeutic agents in combination typically
is carried out over a defined time period (for example, minutes,
hours, days, or weeks, depending upon the combination
selected).
[0445] Administered "in combination" or "co-administration," as
used herein, means that two or more different treatments are
delivered to a subject during the subject's affliction with a
medical condition (e.g., cancer or a neoplastic disorder), in any
order. For example, in some embodiments, the two or more treatments
are delivered after the subject has been diagnosed with a disease
or disorder, and before the disease or disorder has been cured or
eliminated, or when a subject is identified as being at risk but
before the subject has developed symptoms of the disease. In some
embodiments, the delivery of one treatment is still occurring when
the delivery of the second treatment begins, so that there is
overlap. In some embodiments, the first and second treatment are
initiated at the same time. These types of delivery are sometimes
referred to herein as "simultaneous," "concurrent," or
"concomitant" delivery. In other embodiments, the delivery of one
treatment ends before delivery of the second treatment begins. This
type of delivery is sometimes referred to herein as "successive" or
"sequential" delivery.
[0446] In some embodiments, the two treatments (e.g., at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, and a checkpoint inhibitor) are comprised
in the same composition. Such compositions may be administered in
any appropriate form and by any suitable route. In other
embodiments, the two treatments (e.g., at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and a checkpoint inhibitor) are administered in separate
compositions, in any appropriate form and by any suitable route.
For example, in some embodiments, a composition comprising a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and a composition comprising a checkpoint inhibitor may
be administered concurrently or sequentially, in any order at
different points in time; in either case, they should be
administered sufficiently close in time so as to provide the
desired therapeutic or prophylactic effect.
[0447] In embodiments of either simultaneous or sequential
delivery, treatment may be more effective because of combined
administration. In some embodiments, the first treatment is more
effective, e.g., an equivalent effect is seen with less of the
first treatment (e.g., with a lower dose), than would be seen if
the first treatment were administered in the absence of the second
treatment. In some embodiments, the first treatment is more
effective such that the reduction in a symptom, or other parameter
associated with the disease or disorder, is greater than what would
be observed with the first treatment delivered in the absence of
the second treatment. In other embodiments, an analogous situation
is observed with the second treatment. In some embodiments, the
benefit of combination therapy (e.g., the effect on at least one
symptom or the risk/rate of disease progression) is additive. In
some embodiments, the benefit of combination therapy is
superadditive.
[0448] In some embodiments, the present disclosure provides a
method of treating cancer in a subject in need thereof and/or a
subject having or suspected of having a neoplastic disorder by
administering to the subject a therapeutically effective amount of
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing; and at least one
additional therapy (e.g., a checkpoint inhibitor therapy, a
cytokine or cytokine analog, a neoantigen vaccine, CAR-T). In some
embodiments, administration of at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, induces at least one neoantigen and/or a T-cell
response. In some embodiments, administration of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, induces
a double-stranded RNA immune response. In some embodiments,
administration of a therapeutically effective amount of at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, induces immunogenic cell
death. In some embodiments, the at least one additional therapy may
comprise at least one, at least two, at least three, at least four,
or at least five additional therapies. For example, in some
embodiments, a therapeutically effective amount of at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, may be administered in combination with
two checkpoint therapies, i.e., using two different checkpoint
inhibitors. In some embodiments, at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, may be administered in combination with a checkpoint
inhibitor therapy and a neoantigen vaccine.
[0449] In some embodiments of combination therapy, the administered
amount of the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and/or
the at least one additional therapy is reduced by 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, relative to a standard
dosage of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and/or
the at least one additional therapy. In some embodiments, the at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, and/or the at least one
additional therapy is administered at least 10%, 15%, 20%, 25%,
30%, 35%, 40%, 45%, 50%, 75%, or 90% less frequently, relative to a
standard dosing regimen of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and/or the at least one additional therapy. In some
embodiments, the administered amount and/or dosage of the at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, and/or the at least one
additional therapy results in lower systemic toxicity and/or
improved tolerance.
[0450] In some embodiments, administration of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, is
initiated before administration of the at least one additional
therapy. In some embodiments, administration of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, is
initiated after administration of the at least one additional
therapy. In some embodiments, administration of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, is
initiated concurrently with administration of the at least one
additional therapy.
[0451] In some embodiments, administration of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, is
repeated at least once after initial administration. In some
embodiments, the amount of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, used for repeated administration is reduced relative to
the amount used for initial administration. In some embodiments,
the amount of the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, used for
repeated administration is reduced relative to a standard dosage of
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, the amount of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, used for repeated administration is reduced by 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 75%, or 90%, relative to a
standard dosage of the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing.
[0452] In some embodiments, administration of the at least one
additional therapy is repeated at least once after initial
administration. In some embodiments, the amount of the at least one
additional therapy used for repeated administration is reduced
relative to the amount used for initial administration. In some
embodiments, the amount of the at least one additional therapy used
for repeated administration is reduced relative to a standard
dosage of the at least one additional therapy. In some embodiments,
the amount of the at least one additional therapy used for repeated
administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%, 40%,
45%, 50%, 75%, or 90%, relative to a standard dosage of the at
least one additional therapy.
[0453] In some embodiments, repeated administration of a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, is concurrent with repeated administration of the at
least one additional therapy. In some embodiments, repeated
administration of a therapeutically effective amount of at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, is sequential or
staggered with repeated administration of the at least one
additional therapy.
[0454] In some embodiments, the present disclosure provides a
method of treating cancer in a subject in need thereof and/or a
subject having or suspected of having a neoplastic disorder by
administering to the subject a therapeutically effective amount of
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing; and a checkpoint
inhibitor therapy. In some embodiments, the checkpoint inhibitor
therapy comprises administering at least one checkpoint inhibitor.
In some embodiments, the subject is intolerant, non-responsive, or
poorly responsive to the at least one checkpoint inhibitor when
administered alone. In some embodiments, a subject may be
considered non-responsive or poorly responsive to the at least one
checkpoint inhibitor as determined using, e.g., the immune-related
Response Criteria (irRC) and/or the immune-related Response
Evaluation Criteria in Solid Tumors (irRECIST). See, e.g., Wolchok
et al. (2009) Clin Cancer Res. 15(23):7412-20; Bohnsack et al.
"Adaptation of the Immune-Related Response Criteria:irRECIST"
(Abstract 4958) ESMO 2014. Exemplary criteria may include those
used in the art to define when tumors in cancer patients improve
("respond"), remain the same ("stabilize"), or worsen ("progress")
during treatment, when the treatment being evaluated is an
immune-oncology drug (e.g., a checkpoint inhibitor). In some
embodiments, a subject may be considered intolerant to the at least
one checkpoint inhibitor if the subject presents with one or more
than one adverse (grade 2+) event identified for the respective
checkpoint inhibitor (e.g., ipilimumab). In some embodiments, for
example, a subject may be considered intolerant to ipilimumab
treatment if the subject presents with one or more adverse events
chosen from enterocolitis, hepatitis, dermatitis (including toxic
epidermal necrolysis), neuropathy, and endocrinopathy (Yervoy.RTM.
(ipilimumab) FDA Label Supplement, 2018).
[0455] In some embodiments, the checkpoint inhibitor is targeted at
PD1/PDL1, CTLA4, OX40, CD40, LAG3, TIM3, GITR, and/or MR. In some
embodiments, the checkpoint inhibitor is targeted at CTLA4, OX40,
CD40, and/or GITR. In some embodiments, the checkpoint inhibitor is
targeted with an inhibitory antibody or other similar inhibitory
molecule. In some other embodiments, the checkpoint inhibitor is
targeted with an agonist antibody or other similar agonist
molecule. In some embodiments, the checkpoint inhibitor comprises a
cytotoxic T-lymphocyte-associated antigen 4 pathway (CTLA4)
inhibitor. In some embodiments, the CTLA4 inhibitor is an
anti-CTLA4 antibody. In some embodiments, the anti-CTLA4 antibody
is ipilimumab. In some embodiments, the checkpoint inhibitor
comprises a programmed death-1 pathway (PD1) inhibitor. In some
embodiments, the PD1 inhibitor is an anti-PD1 antibody. In some
embodiments, the anti-PD1 antibody is nivolumab. In some
embodiments, the PD1 inhibitor is an anti-PDL1 antibody. In some
embodiments, the anti-PDL1 antibody is atezolizumab. In some
embodiments, the checkpoint inhibitor comprises a CTLA4 inhibitor
and a PD1 inhibitor. In some embodiments, the checkpoint inhibitor
is targeted at OX40. In some embodiments, the checkpoint inhibitor
is targeted at CD40. In some embodiments, the checkpoint inhibitor
is targeted at GITR. In some embodiments, the benefit of
combination therapy (e.g., the effect on at least one symptom or
the risk/rate of disease progression) with a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and a
checkpoint inhibitor (e.g., a CTLA4-, PD1/PDL1-, OX40-, CD40-,
and/or GITR-targeted antibody or molecule) is additive. In some
embodiments, the benefit of combination therapy with a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and a checkpoint inhibitor (e.g., a CTLA4-, PD1/PDL1,
OX40-, CD40-, and/or GITR-targeted antibody or molecule) is
superadditive (i.e., synergistic).
[0456] In some embodiments, the present disclosure provides a
method of treating cancer in a subject in need thereof and/or a
subject having or suspected of having a neoplastic disorder by
administering to the subject a therapeutically effective amount of
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing; and a cytokine or
cytokine analog therapy. In some embodiments, the cytokine or
cytokine analog therapy comprises administering at least one
cytokine or cytokine analog. In some embodiments, the subject is
intolerant, non-responsive, or poorly responsive to the at least
one cytokine or cytokine analog when administered alone.
[0457] In some embodiments, the cytokine or cytokine analog
comprises a T-cell enhancer. In some embodiments, the cytokine or
cytokine analog comprises IL-2, IL-10, IL-12, IL-15, IFN.gamma.,
and/or TNF.alpha.. In some embodiments, the cytokine or cytokine
analog comprises IL-2, IL-10, IL-12, and/or IL-15. In some
embodiments, administering the cytokine or cytokine analog enhances
T-cell priming following administration of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, due to
induction and presentation of neoantigens.
[0458] In some embodiments, the cytokine or cytokine analog
comprises IL-2. In some embodiments, IL-2 boosts signals to
effector cells promoting their expansion (Rosenberg (2014) J
Immunol. 192(12):5451-8). In some embodiments, the cytokine or
cytokine analog comprises IL-10. In some embodiments, IL-10 boosts
CD8+ T-cell priming and activation (Mumm et al. (2011) Cancer Cell
20(6):781-96). In some embodiments, the cytokine or cytokine analog
comprises IL-12. In some embodiments, IL-12 links the innate and
adaptive immune responses to boost antigen-specific priming and
targeting (Tugues et al. (2015) Cell Death Differ. 22(2):237-46).
In some embodiments, the cytokine or cytokine analog comprises
IL-15. In some embodiments, IL-15 boosts T-effector (CD8) cell
priming and/or activation. In some embodiments, the cytokine or
cytokine analog comprises IFN.gamma.. In some embodiments,
IFN.gamma. supplements T-effector cell secretion of IFN.gamma.. In
some embodiments, the cytokine or cytokine analog comprises
TNF.alpha.. In some embodiments, TNF.alpha. supplements T-effector
cell secretion of TNF.alpha..
[0459] In some embodiments, an initial dose of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, is
administered to a subject to trigger aberrant splicing and
production of neoantigen peptides. After a period of time to allow
for protein production and antigen presentation, in some
embodiments, the subject is then administered an initial dose of a
cytokine or cytokine analog to boost and/or enhance effector T-cell
priming and expansion. In some embodiments, the wait period between
doses of the at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and
cytokine or cytokine analog is about 2, about 3, about 4, about 5,
about 6, or about 7 days. In some embodiments, the wait period is
between about 3 days and about 5 days. In some embodiments, the
cytokine or cytokine analog is IL-2, IL-10, IL-12, IL-15,
IFN.gamma., and/or TNF.alpha.. In some embodiments, the combination
therapeutic benefit of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing, and a cytokine or cytokine analog may be additive or
superadditive.
[0460] In some embodiments, after a period to allow for T-cell
priming and expansion, the subject is then administered a second or
subsequent dose of a therapeutically effective amount of at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, to trigger
re-presentation of neoantigen peptides. In some embodiments, the
wait period between an initial dose of a cytokine or cytokine
analog and a second or subsequent dose of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, is about
2, about 3, about 4, or about 5 weeks. In some embodiments, the
wait period is about 3 weeks. In some embodiments, subsequent doses
of the cytokine or cytokine analog may be administered, e.g.,
interspersed between subsequent doses of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing.
Following a second or subsequent dose of a therapeutically
effective amount of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, in some
embodiments, the immune system may engage with the
neoantigen-presenting tumor cells and/or elicit tumor cell killing.
In some embodiments, dosing of a therapeutically effective amount
of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing,
following this exemplary initial treatment regimen can be
pulsatile, i.e., the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, may be
dosed at prolonged intervals (e.g., about every 4 weeks, about
every 5 weeks, about every 6 weeks) to allow for antigen
presentation, T-cell engagement and/or tumor cell killing, and/or
recovery of the memory T-cell population.
[0461] In some embodiments, the subject has a non-synonymous
mutational burden of about 150 mutations or less. In some
embodiments, the subject has a non-synonymous mutational burden of
about 100 mutations or less. In some embodiments, the subject has a
non-synonymous mutational burden of about 50 mutations or less. In
some embodiments, the subject has or is suspected of having a
neoplastic disorder, e.g., a hematological malignancy or a solid
tumor. In some embodiments, the hematological malignancy is chosen
from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In
some embodiments, the hematological malignancy is chosen from acute
myeloid leukemia and multiple myeloma. In some embodiments, the
solid tumor is chosen from breast cancer, gastric cancer, prostate
cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct
carcinoma, melanoma, colon cancer, and esophageal cancer. In some
embodiments, the solid tumor is chosen from HER2-positive breast
cancer, gastric adenocarcinoma, and prostate cancer.
[0462] In some embodiments, the subject is in need of a method of
treating cancer. In some embodiments, the cancer is a hematological
malignancy or a solid tumor. In some embodiments, the hematological
malignancy is chosen from a B-cell malignancy, a leukemia, a
lymphoma, and a myeloma. In some embodiments, the hematological
malignancy is chosen from acute myeloid leukemia and multiple
myeloma. In some embodiments, the solid tumor is chosen from breast
cancer, gastric cancer, prostate cancer, ovarian cancer, lung
cancer, uterine cancer, salivary duct carcinoma, melanoma, colon
cancer, and esophageal cancer. In some embodiments, the solid tumor
is chosen from HER2-positive breast cancer, gastric adenocarcinoma,
and prostate cancer.
[0463] In some embodiments, a patient having a cancer as described
herein can be treated with a combination of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing and a neoantigen vaccine. Without being bound
by theory, vaccines, used alone or in combination with immune
checkpoint inhibitor (ICI) molecules, have shown promise in early
trials (Ott et al. (2017) Nature 547(7662):217-21; Sahin et al.
(2017) Nature 547(7662):222-6), but generally require sequencing of
patient tumor mutations (Ott et al. (2017) Nature 547(7662):217-21;
Aldous and Dong (2018) Bioorg. Med. Chem. 26(10):2842-9). As such,
vaccines are often dependent on sufficient numbers of
non-synonymous mutations that are antigenic. In general, tumors
with very low mutation burden provide few candidate antigens, and
those with rapid growth provide limited time to identify and
produce patient-specific vaccines.
[0464] To date, attempts to develop vaccines that would be broadly
immunogenic across a large percentage of patients have focused on
proteins that are either frequently mutated, ectopically
overexpressed, or amplified, and/or that exist as "self" proteins
within the organism. In addition, these proteins are often
expressed in immunologically restricted tissues (e.g., neuronal
markers expressed in neuroendocrine tumor types), while others may
be normally expressed during embryogenesis (e.g., oncofetal
antigens). Thus, utility of vaccines using such proteins as
antigens is often limited to specific tumor lineages or subsets
where one or more of the antigens are presented. Vaccine utility
would also need to be confirmed by sequencing of patient tumor
samples, which can be time-consuming.
[0465] Moreover, if these antigens exist as "self" proteins, the
immune system would likely be primed to recognize these as "self"
and thus, not respond. Or, alternatively, if the immune system is
able to mount an effector response to these antigens, it may lead
to on-target side effects in tissues where the antigen may be
expressed. In both of these cases, one of the key challenges is
that most antigenic peptides are derived from "passenger" genes
(i.e., genes that are mutated or amplified in the course of
tumorigenesis, but that do not play a critical role in the
continued survival or proliferation of the tumor itself). As such,
these genes may be silenced without significant consequence to the
tumor progression, and thus would allow a tumor to "escape" an
immune response against these antigens. Without wishing to be bound
by theory, this mechanism may play a role in tumor evolution, where
random mutations that are strongly antigenic are often "selected
against" by the tumor during the early stages of tumorigenesis
(Dunn et al. (2004) Annu. Rev. Immunol. 22:329-60).
[0466] In addition, certain evidence also indicates that chronic
antigen presentation and immune stimulation may lead to immune cell
anergy and exhaustion (Pardoll (2012) Nat. Rev. Cancer
12(4):252-64). These phenotypes underlie the therapeutic rationale
behind current ICI treatments, as ICI has been shown to either
repress the exhausted immune cell phenotype (.alpha.-PD1/PD-L1) or
to facilitate additional immune cell responses (.alpha.-CTLA4).
Notably, with .alpha.-CTLA4 therapy, a certain subset of patients
have been reported to exhibit severe immune-related adverse events
that may be ascribed to the promotion of T-cell activation and a
break of the immune tolerance mechanisms that restrain
self-reactive immune responses.
[0467] Both of these approaches (i.e., triggering or enhancing de
novo immune responses to neoantigens or derepressing the anergy or
exhaustion of existing immune responses) are linked to a chronic
immune activation. As such, these approaches are sensitive to
anergy, editing, and other tumor-mediated mechanisms designed to
suppress immune engagement.
[0468] In contrast, treatment with at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing may induce an immune response to novel sequences
representing neoantigens. In some embodiments, presentation of
neoantigens provides the adaptive immune system with more divergent
targets with which to engage and activate. In some embodiments, the
ability of at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing to
acutely induce alternative splicing and the resulting neoantigens
may reduce the risk of immune system fatigue due to chronic
exposure to mutation-driven neoantigens and/or limit the ability of
tumor cells to adapt to evade therapy. In some embodiments,
administering at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing in
combination with a neoantigen vaccine enhances the immune response
to the neoantigens produced by the at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing. In some embodiments, the at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing is administered before, during, or after vaccination. In
some embodiments, the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing
and/or vaccine may be administered once or more than once during
the course of treatment. In some embodiments, the vaccine is
administered once and the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing is administered more than once during the course of
treatment. In some embodiments, the vaccine is administered once
and then one or more boosters are administered during the course of
treatment.
[0469] As used herein, the term "neoantigen vaccine" refers to a
pooled sample of one or more immunogenic neoantigen peptides or
mRNAs, for example at least two, at least three, at least four, at
least five, or more neoantigen peptides. The term "vaccine" refers
to a composition for generating immunity for the prophylaxis and/or
treatment of a disease (e.g., a neoplastic disorder, e.g., a
hematological malignancy or solid tumor). Accordingly, vaccines are
medicaments which comprise immunogenic agents and are intended to
be used in humans or animals for generating specific immune
defenses and protective substances after vaccination. A neoantigen
vaccine can additionally include a pharmaceutically acceptable
carrier, diluent, excipient, and/or adjuvant.
[0470] As used herein, the term "immunogenic" refers to any agent
or composition that can elicit an immune response, e.g., a T-cell
response. The immune response can be antibody- or cell-mediated, or
both.
[0471] In some embodiments, a patient is given at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing and then given a peptide or mRNA vaccine of
known neoantigen to enhance immune response to the neoantigens
produced by the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
other embodiments, a patient is given at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing and screened for neoantigens produced by the treatment.
Subsequently, one or more of those neoantigens are used to create a
personalized vaccine that is given to the patient. In either of
these embodiments, the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing
and/or peptide or mRNA vaccine may be administered to the patient
once or repeatedly.
[0472] In some embodiments, a suitable neoantigen for a vaccine can
be identified by screening a panel of transcripts with altered
splicing and robust expression from one or more tissue samples in a
patient (e.g., from a tumor biopsy). In some embodiments, variant
protein sequences are identified in the screened sample based on
translation across the aberrantly spliced mRNA junction while
retaining portions of the protein sequence (up to 12 amino acids)
flanking the junction-spanning amino acid changes. In some
embodiments, these junction-spanning peptide fragments are scanned
for high affinity binding to MHC1 alleles, e.g., using a tool such
as NetMHC1 (Nielsen et al. (2003) Protein Sci 12(5):1007-17;
Andreatta and Neilsen (2016) Bioinformatics 32(4):511-7). These
results allow for filtering of the neopeptides to those that are
predicted high affinity binders for a unique patient HLA allele
makeup as well as assembly of pools of neopeptides predicted to be
broadly binding to HLA alleles that are present with high
frequencies in different populations (Maiers et al. (2007) Hum
Immunol 68(9):779-88). In some embodiments, the identified
neopeptides are then formulated as a vaccine, e.g., by conjugation
to a suitable carrier or adjuvant (Ott et al. (2017) Nature
547(7662):217-21), or for delivery as an mRNA (Sahin et al. (2017)
Nature 547(7662):222-6).
[0473] In some embodiments, the selected neoantigen is based on a
screen of an individual patent's tumor response to the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing to identify one or more neoantigens
resulting from treatment to use in subsequent vaccination. In other
embodiments, a neoantigen is chosen, e.g., based on screening a
panel of samples from different patients to identify common
neoantigens produced by the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing and then used as a universal vaccine for future
patients.
[0474] Without being bound by theory, in some embodiments, use of a
universal neoantigen vaccine would avoid the need to sequence and
analyze the unique mutation status of each patient's tumor because
the chosen neoantigens are not dependent on tumor mutation but
rather mimic a neoantigen produced by at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing and generally recognized by the body as foreign. In
addition, in some embodiments, use of a neoantigen vaccine may be
particularly effective since a patient's tumor cells may be more
likely to mutate away from producing one or more neoantigens that
are dependent on tumor mutation, as compared to those that mimic a
neoantigen produced by at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing. This
may allow for the formulation of a bulk vaccine that would be
broadly immunogenic across a large percentage of patients,
expediting the initiation of a treatment regime. Patients may be
vaccinated according to the schedules outlined herein and, prior to
following completion of the vaccination, could be further treated
with at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, e.g., to
induce expression of the neoantigen peptides. In some embodiments,
patients may be administered at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing before, at the same time as, or after vaccination. In
some embodiments, patients are administered at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing, screened for one or more neoantigens found in
a panel of universal neoantigens, and vaccinated with a universal
neoantigen vaccine comprising at least one universal neoantigen
identified in the subject. In some embodiments, patients may be
administered at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing once or
more than once after vaccination. The at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing and/or the vaccine may be administered once or more than
once during the course of treatment.
[0475] In some embodiments, a vaccine may comprise one or more than
one neoantigen peptide or mRNA. In some embodiments, a vaccine may
comprise one or more than one long neoantigen peptide. Such "long"
neoantigen peptides, in some embodiments, undergo efficient
internalization, processing, and cross-presentation in professional
antigen-presenting cells such as dendritic cells. Similarly, long
vaccine peptides have been shown, in other contexts, to induce
cytotoxic T-cells in humans (Melief and van der Burg (2008) Nat Rev
Cancer 8(5):351-60). In some embodiments, a neoantigen peptide is
extended to comprise the neoantigen peptide sequence itself in
addition to flanking amino acid sequences. In some embodiments, the
extended peptide sequence facilitates the uptake of protein by
antigen-presenting cells, e.g., dendritic cells. In some
embodiments, the extended peptide sequence enables efficient
antigen presentation and T-cell priming in models with different
HLA isotypes. In some embodiments, a longer neoantigen peptide
and/or extended peptide sequence exhibits increased uptake by
antigen-presenting cells (e.g., dendritic cells), increased antigen
presentation, and/or increased T-cell priming, as compared to a
shorter neoantigen peptide and/or shorter peptide sequence (e.g., a
peptide sequence less than about 10 or less than about 5 amino
acids in length). In some embodiments, a long neoantigen peptide
ranges from about 5 to about 50 amino acids in length. In some
embodiments, a long neoantigen peptide ranges from about 10 to
about 50 amino acids in length. In some embodiments, the at least
one neoantigen peptide ranges from about 10 to about 35 amino acids
in length. In some embodiments, a long neoantigen peptide ranges
from about 15 to about 25 amino acids in length.
[0476] In some embodiments, the neoantigen sequence and/or
antigenic portion ranges from about 10 to about 35 amino acids in
length. In some embodiments, the neoantigen sequence and/or
antigenic portion ranges from about 15 to about 25 amino acids in
length. In some embodiments, the neoantigen sequence and/or
antigenic portion ranges from about 10 to about 20 amino acids in
length. In some embodiments, the neoantigen sequence and/or
antigenic portion does not exclusively overlap or consist of the
canonical peptide sequence (e.g., any of the exemplary canonical
peptide sequences underlined in Table 13).
[0477] Amino acid sequences of exemplary long neoantigen peptides
are set forth in Table 13.
[0478] These exemplary neoantigen peptides are generated after
administration of ADCs containing pladienolide splicing modulators,
however, given the similar mechanism of action (i.e., similar
mechanisms of splicing modulation), similar neoantigen peptides may
be produced by compounds chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing.
TABLE-US-00002 TABLE 12 Neopeptides SEQ ID Event Observed
Neopeptide NO Junction (HG19) Gene type in 1 SPTLPPRSL 1 chr12:
49663470- TUBA1C Intron H1568 49663610: + retention 2 HPSIKRGLSSL 2
chr12: 42729776- PPHLN1 Exon H1568 42781257: + skipping 3 LLLPHHVL
3 chr12: 49663470- TUBA1C Intron H1568 49663610: + retention 4
RTAPGVRPPF 4 chr14: 35182767- CFL2 Intron H1568 35183743: -
retention 5 RPQKSIQAL 5 chr10: 28822963- WAC Intron H1568 28823162:
+ retention 6 APAPPPLPA 6 chr17: 80009840- GPS1 Intron H1568
80011149: + retention 7 RPRPSFPVSL 7 chr7: 55087058- EGFR Intron
H1568 55134942: + retention 8 RPKHGDGFSL 8 chr11: 57472287- MED19
Intron H1568 57472444: - retention 9 GPAPGKTGL 9 chr7: 75932393-
HSBP1 Intron H1568 75933118: + retention 10 EAARKGNSL 10 chr1:
53480715- SCP2 Exon H1568 53504588: + skipping 11 RIKEKIEEL 11
chr9: 72897499- SMC5 Exon H1568 72912881: + skipping 12 EIKKRFRQF
12 chr1: 28531860- DNAJC8 Exon H1568 28541450: - skipping 13
HESAAMAET 13 chr11: 102272937- TMEM123 Exon HCC1954 102323254: -
skipping 14 ALKLKQVGV 14 chr1: 153610924- CHTOP Exon H1568
153617539: + skipping 15 DLKKRHITF 15 chr13: 41323417- MRPS31 Exon
H1568 41331008: - skipping 16 DVKRNDIAM 16 chr1: 41213277- NFYC
Exon H1568 41218822: + skipping 17 IPSDHILTPA 17 chr6: 149718900-
TAB2 Exon H1568 149720239: + skipping 18 TVFSTSSLK 18 chr11:
61197654- SDHAF2 Exon H1568 61213412: + skipping 19 ITSCLLNF 19
chr5: 137892555- HSPA9 Intron H1568 137893090: - retention 20
RASPVRGQL 20 chr7: 75677544- MDH2 Intron H1568 75677893: +
retention 21 VVRKPVIAL 21 chr1: 36923582- MRPS15 Exon H1568
36929406: - skipping 22 LLSEKKKIS 22 chr6: 31750622- VARS Intron
H1568 31750872: - retention 23 APASKPRPRL 23 chr19: 3573798- HMG20B
Intron H1568 3574380: + retention 24 RYGQLSEKF 24 chr19: 33076813-
PDCD5 Exon HCC1954 33078158: + skipping 25 VYISNVSKL 25 chr3:
53920961- SELK Exon HCC1954 53925796: - skipping 26 LPTKETPSF 26
chr2: 85133241- TMSB10 Alt 3'ss HCC1954 85133394: + 27 GEAPPPPPA 27
chr17: 80223672- CSNK1D Intron HCC1954 80231181: - retention 28
LEEISKQEI 28 chr17: 27804724- TAOK1 Exon HCC1954 27807385: +
skipping 29 IYNHITVKI 29 chr4: 2886393- ADD1 Exon HCC1954 2896308:
+ skipping
[0479] The protein sequences of the twenty nine neopeptides listed
in Table 12 can be extended. The extended protein sequence
incorporates both the neopeptide sequence itself in addition to
flanking amino acid sequences. The extended protein sequence better
facilitates the uptake of protein by dendritic cells and enables
antigen presentation and T-cell priming in models with different
HLA isotypes. Amino acid sequences of the twenty nine extended
neopeptides are set forth in Table 13.
TABLE-US-00003 TABLE 13 Amino acid sequences of extended
neopeptides SEQ Extended neopeptide amino Gene ID NO acid sequence*
TUBA1C 30 VDLEPTVIGELTSVTQVRSQGAGTGG LSWGGSAGHSPTLPPRSLSLLLLPHH
VLQMKFALALTASSSTLNSSQARKM LPITMPEGTTPLARRSLTSCWTEFAS
WLTSAPVFRASWFSTALVGELVLGSP RCSWNVSQLIMARSPSWSSPFTRRPR FPQL PPHLN1
31 APPRSHPSIKRGLSSL CFL2 32 MVRRARWPGGRGEARKAPRTAPGVRP PF WAC 33
WVNCLFVSGRAAAGGGGGGAVPPYLE LAGPPFLLLTLIRIGLGRRSGRAGGR
AGTQCGGERGPGFAAFRPLRPFRRLR VCAVCVRGSALGRSVGLPRGGAAGAP
FSSSPAPHPRRVLCRCLLFLFFSCHD RRGDSQPYQVPAEAGVEGLEGAGGGR
EGLLLERRPQKSIQALRCNTSETSTA DPLKIPGLVPLALSSKV GPS1 34
MPLPVQVFNLQVTSRGRPGPPRPRAP RHWGRAEVEQGRGACARSRSGTLRAG
PPRAARVGGCRAEGASPPWLRAAIGG RRAAPAPPPLPAAHGRGSRPPRR EGFR 35
QPAQPRTGAPARRPRPRPSFPVSLRS AAPPTGTAGGTGRFVLRPGESGAGGG
GDAWDTGLQARRGTAAGTSGAPNRSQ LSSLTFPAQLRRIGVSGRKPGAGGRL
GPGSRTCAPRCLPRARRGPGAHPRGG RCPPAETALFREAEEGTQKYSLPSDP AGQAAF MED19
36 FRLHTGPVSPVGGRRQMGRPKHGDGF SLQVCSFIMEQNG HSBP1 37
GVVEITGEPPCSCRGEEEASRAGRAG GVRLKRGSRGPGELNVGPAPGKTGLL
IPLLRNWECGSLLRALSAL SCP2 38 KMGFPEAARKGNSL SMC5 39
LEARIKEKIEELQQALI DNAJC8 40 EIKKRFRQFKQAVYKQ TMEM123 41
AHESAAMAETLQHVPS CHTOP 42 NRPSVQAALKLKQVGV MRPS31 43
KTDDLKKRHITFTLGCGIC NFYC 44 MKLDEDVKRNDIAMAI TAB2 45
NSISQIPSDHILTPALFITFMTILDL SDHAF2 46 TVFSTSSLKLNQPQKYLKMKSWPC HSPA9
47 AEEDRRKKVITSCLLNFNLSKAQS MDH2 48 RSFSTSAQVGQTRGGLQAEAPRPGPR
ASPVRGQL MRPS15 49 RGYVVRKPVIALSVKI VARS 50
VDMDFGTGGQGAGPVGRGKDWSCTLA VHLLSEKKKISFSQIDRAWGGSQGTV
LDKWGPGVVSELHPSAKEVSVGRNSV ESLMTWAS HMG20B 51
EKGSHEEEVRVPALSWGRPRAPAPAS KPRPRLDLNCLWLRPQPIFLWKLRPR
PVPAATPLTGPLPL PDCD5 52 RYGQLSEKFNRRKVMDS SELK 53 MVYISNVSKLCFSKM
TMSB10 54 NTLPTKETPSFLLNPHTSWVPRPHRE APRLRVGVAAPLQRPLPALHSH CSNK1D
55 FGDIYLGEAPPPPPAARRPGPCGCQD QARSRKEVVAPAGSPRKSRHRRIVAR TQRPLG
TAOK1 56 GSASDLLEEISKQEISF ADD1 57 QLIYNHITVKINLQGD *Underline
indicates amino acids derived from the canonical transcript reading
open frame (i.e., the canonical peptide sequence).
[0480] As used herein, a neoantigen peptide or mRNA vaccine
encompasses using a fragment of a neoantigen peptide or its
encoding mRNA, so long as that fragment retains immunogenic
potential.
[0481] In some embodiments, a neoantigen vaccine comprises at least
one neoantigen peptide. In some embodiments, a neoantigen vaccine
comprises at least 2, at least 3, at least 4, at least 5, at least
6, at least 7, at least 8, at least 9, at least 10, at least 12, at
least 15, or at least 20 neoantigen peptides. In some embodiments,
the neoantigen peptide(s) range from about 5 to about 50 amino
acids in length. In some embodiments, the neoantigen peptide(s)
range from about 10 to about 50 amino acids in length. In some
embodiments, the at least one neoantigen peptide ranges from about
10 to about 35 amino acids in length. In some embodiments, the
neoantigen peptide(s) range from about 15 to about 25 amino acids
in length.
[0482] In some embodiments, the present disclosure provides a
method of treating a subject having or suspected of having a
neoplastic disorder by administering to the subject a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing; and a neoantigen vaccine. A neoantigen vaccine may be,
e.g., a peptide or mRNA neoantigen vaccine. In some embodiments,
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing is
administered before administration of the neoantigen vaccine. In
some embodiments, the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing is
administered after administration of the neoantigen vaccine. In
some embodiments, the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing is
administered concurrently with administration of the neoantigen
vaccine. In some embodiments, administration of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing is repeated at least once after initial
administration. In some embodiments, the amount of the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing used for repeated administration is reduced
as compared to the amount used for initial administration.
[0483] In some embodiments, the present disclosure further provides
a combination comprising at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing; and a neoantigen vaccine (e.g., a universal neoantigen
vaccine) for use in treating a subject having or suspected of
having a neoplastic disorder. In some embodiments, the neoantigen
vaccine is a peptide or mRNA neoantigen vaccine. In some
embodiments, the combination further comprises at least one
additional therapy. In some embodiments, the at least one
additional therapy comprises at least one, at least two, at least
three, at least four, or at least five additional therapies.
[0484] In some embodiments, the present disclosure further provides
a method of treating a subject having or suspected of having a
neoplastic disorder by (a) administering to the subject a
therapeutically effective amount of at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing; (b) detecting one or more neoantigens in the subject
after administration of the at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing; (c) comparing the one or more neoantigens to a panel of
universal neoantigens; and (d) administering to the subject a
universal neoantigen vaccine comprising at least one universal
neoantigen present in the subject. In some embodiments, the
universal neoantigen vaccine is administered alone or in
combination with at least one additional therapy. In some
embodiments, the at least one additional therapy comprises at least
one, at least two, at least three, at least four, or at least five
additional therapies.
[0485] In some embodiments, the at least one additional therapy
comprises repeated administration of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. In some embodiments, repeated administration
of the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing is
initiated before administration of the universal neoantigen
vaccine. In some embodiments, repeated of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing is initiated after administration of the
universal neoantigen vaccine. In some embodiments, repeated
administration of the at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing is
initiated concurrently with administration of the universal
neoantigen vaccine. In some embodiments, the amount of the at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing used for repeated
administration is reduced as compared to the amount used for
initial administration. In some embodiments, the amount of the at
least one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing used for the initial
and/or repeated administration is reduced as compared to a standard
dosage of the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing when used
without a vaccine treatment. In some embodiments, the amount of the
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing used for initial and/or
repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage of the
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing.
[0486] In some embodiments, the at least one additional therapy
comprises administering a checkpoint inhibitor (e.g., any of the
exemplary checkpoint inhibitors described herein). In some
embodiments, administration of the checkpoint inhibitor is
initiated before administration of the universal neoantigen vaccine
and/or repeated administration of the at least one compound chosen
from compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing. In some embodiments, administration of the checkpoint
inhibitor is initiated after administration of the universal
neoantigen vaccine and/or repeated of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. In some embodiments, administration of the
checkpoint inhibitor is initiated concurrently with administration
of the universal neoantigen vaccine and/or repeated administration
of the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, administration of the checkpoint inhibitor is repeated
at least once after initial administration. In some embodiments,
the amount of the checkpoint inhibitor used for repeated
administration is reduced as compared to the amount used for
initial administration. In some embodiments, the amount of the
checkpoint inhibitor used for repeated administration is reduced as
compared to a standard dosage of the checkpoint inhibitor. In some
embodiments, the amount of the checkpoint inhibitor used for
repeated administration is reduced by 10%, 15%, 20%, 25%, 30%, 35%,
40%, 45%, 50%, 75%, or 90%, as compared to a standard dosage of the
checkpoint inhibitor. In some embodiments, the subject is
intolerant, non-responsive, or poorly responsive to the checkpoint
inhibitor when administered alone.
[0487] Also provided herein, in some embodiments, are neoantigen
vaccines comprising at least one neoantigen peptide or at least one
neoantigen mRNA. In some embodiments, a neoantigen vaccine
comprises at least one neoantigen peptide. In some other
embodiments, a neoantigen vaccine comprises at least one neoantigen
mRNA.
[0488] Also provided herein, in some embodiments, are kits
comprising at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing; and a
neoantigen vaccine (e.g., a universal neoantigen vaccine). In some
embodiments, the neoantigen vaccine is a peptide or mRNA neoantigen
vaccine. In some embodiments, the kit further comprises one or more
additional components, including but not limited to: instructions
for use; other agents, e.g., one or more additional therapeutic
agents; devices, containers, or other materials for preparing the
at least one compound chosen from compounds of Formula I, compounds
of Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, and/or neoantigen vaccine
for therapeutic administration; pharmaceutically acceptable
carriers; and devices, containers, or other materials for
administering the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and/or
neoantigen vaccine to a patient. Instructions for use can include
guidance for therapeutic applications including suggested dosages
and/or modes of administration, e.g., in a patient having or
suspected of having a neoplastic disorder. In some embodiments, the
kit further contains instructions for therapeutic use, e.g., use of
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and the
neoantigen vaccine to treat or prevent a neoplastic disorder in a
patient. In some embodiments, the kit further contains at least one
additional therapeutic agent (e.g., for administering together with
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and the
neoantigen vaccine, e.g., a checkpoint inhibitor). In some
embodiments, the at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, and/or
neoantigen vaccine is formulated as a pharmaceutical
composition.
[0489] In some embodiments of the methods and compositions
disclosed herein, the neoantigen vaccine comprises at least one
neoantigen peptide. In some embodiments, the at least one
neoantigen peptide ranges from about 10 to about 50 amino acids in
length. In some embodiments, the at least one neoantigen peptide
ranges from about 10 to about 35 amino acids in length. In some
embodiments, the at least one neoantigen peptide ranges from about
15 to about 25 amino acids in length.
[0490] In some embodiments, the at least one neoantigen peptide
comprises one or more than one neoantigen sequence disclosed
herein.
[0491] In some embodiments, the neoantigen sequence and/or
antigenic portion ranges from about 10 to about 35 amino acids in
length. In some embodiments, the neoantigen sequence and/or
antigenic portion ranges from about 15 to about 25 amino acids in
length. In some embodiments, the neoantigen sequence and/or
antigenic portion ranges from about 10 to about 20 amino acids in
length. In some embodiments, the neoantigen sequence and/or
antigenic portion does not exclusively overlap or consist of the
canonical peptide sequence (e.g., any of the exemplary canonical
peptide sequences underlined in Table 13).
[0492] In some embodiments, the neoantigen sequence is a neoantigen
sequence specific to the subject. In some embodiments, the
neoantigen sequence is a personalized neoantigen vaccine for the
subject. In some embodiments, the neoantigen sequence is capable of
binding to at least one HLA allele expressed in the subject.
[0493] In some other embodiments, the neoantigen sequence is a
universal neoantigen sequence. In some embodiments, the neoantigen
sequence is a universal neoantigen vaccine. In some embodiments,
the neoantigen sequence is capable of binding to at least one HLA
allele expressed in at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% of subjects in a population of subjects suffering from the
neoplastic disorder. In some embodiments, the neoantigen sequence
is capable of eliciting a T-cell response against a tumor present
in at least 1%, at least 5%, or at least 10% of a population of
subjects suffering from the neoplastic disorder.
[0494] In some embodiments, the neoantigen sequence has been
identified by sequencing at least one neoantigen peptide induced in
the subject by administering a therapeutically effective amount of
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, the at least one neoantigen peptide comprises a
neoantigen sequence induced by contacting a neoplastic cell with a
therapeutically effective amount of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. In some embodiments, the neoplastic cell is
present in an in vitro cell culture. In some embodiments, the
neoplastic cell is obtained from the subject. In some embodiments,
the neoplastic cell is present in the subject.
[0495] In some embodiments, the neoantigen vaccine comprises at
least one neoantigen peptide or mRNA and a pharmaceutically
acceptable carrier. In some embodiments, a neoantigen peptide or
mRNA can be linked to a suitable carrier to help elicit an immune
response. Exemplary carriers for linking to immunogenic agents
(e.g., a neoantigen peptide or mRNA) include serum albumins,
keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin,
ovalbumin, tetanus toxoid, or a toxoid from other pathogenic
bacteria, such as diphtheria, E. coli, cholera, or H. pylori, or an
attenuated toxin derivative. Other carriers for stimulating or
enhancing an immune response include cytokines such as IL-1,
IL-1.alpha. and .beta. peptides, IL-2, .gamma.INF, IL-10, GM-CSF,
and chemokines, such as M1P1.alpha. and .beta. and RANTES.
Immunogenic agents can also be linked to peptides that enhance
transport across tissues, as described, e.g., in WO 97/17613 and WO
97/17614. In some embodiments, the pharmaceutically acceptable
carrier is chosen from a peptide, a serum albumin, a keyhole limpet
hemocyanin, an immunoglobulin, a thyroglobulin, an ovalbumin, a
toxoid or an attenuated toxoid derivative, a cytokine, and a
chemokine.
[0496] In some embodiments, the neoantigen peptide or mRNA may be
linked to the pharmaceutically acceptable carrier. Immunogenic
agents can be linked to carriers by chemical crosslinking.
Techniques for linking an immunogenic peptide to a carrier include
the formation of disulfide linkages using
N-succinimidyl-3-(2-pyridyl-thio) propionate (SPDP) and
succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)
(if the peptide lacks a sulfhydryl group, this can be provided by
addition of a cysteine residue). These reagents create a disulfide
linkage between themselves and peptide cysteine resides on one
protein and an amide linkage through the epsilon-amino on a lysine,
or other free amino group in other amino acids. A variety of such
disulfide/amide-forming agents are described in Jansen et al.
((1982) Immun Rev. 62:185). Other bifunctional coupling agents form
a thioether rather than a disulfide linkage. Many of these
thioether-forming agents are commercially available and include
reactive esters of 6-maleimidocaproic acid, 2-bromoacetic acid, and
2-iodoacetic acid, 4-(N-maleimido-methyl)cyclohexane-1-carboxylic
acid. The carboxyl groups can be activated by combining them with
succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. In
some embodiments, the neoantigen peptide and the pharmaceutically
acceptable carrier are covalently attached via a linker.
[0497] Neoantigen and other such immunogenic peptides can also be
expressed as fusion proteins with carriers. The immunogenic peptide
can be linked at the amino terminus, the carboxyl terminus, or at a
site anywhere within the peptide (internally) to the carrier. In
some embodiments, multiple repeats of the immunogenic peptide can
be present in the fusion protein. In some embodiments, the
neoantigen peptide and the pharmaceutically acceptable carrier are
expressed as a fusion protein.
[0498] In some embodiments, the neoantigen vaccine comprises at
least one neoantigen peptide or its encoding mRNA and a
pharmaceutically acceptable diluent. In some embodiments, the
neoantigen vaccine comprises at least one neoantigen peptide or its
encoding mRNA and a pharmaceutically acceptable adjuvant (e.g., an
adjuvant as described herein).
[0499] In some embodiments of the methods and compositions
disclosed herein, the neoantigen vaccine comprises at least one
neoantigen mRNA. In some embodiments, the at least one neoantigen
mRNA encodes one or more than one neoantigen sequence.
[0500] In some embodiments, the neoantigen sequence and/or
antigenic portion ranges from about 10 to about 50 amino acids in
length. In some embodiments, the at least one neoantigen peptide
ranges from about 10 to about 35 amino acids in length. In some
embodiments, the neoantigen sequence and/or antigenic portion
ranges from about 15 to about 25 amino acids in length. In some
embodiments, the neoantigen sequence and/or antigenic portion
ranges from about 10 to about 20 amino acids in length. In some
embodiments, the neoantigen sequence and/or antigenic portion does
not exclusively overlap or consist of the canonical peptide
sequence (e.g., any of the exemplary canonical peptide sequences
underlined in Table 13).
[0501] In some embodiments, the neoantigen sequence is a neoantigen
sequence specific to the subject. In some embodiments, the
neoantigen sequence is a personalized neoantigen vaccine for the
subject. In some embodiments, the neoantigen sequence is capable of
binding to at least one HLA allele expressed in the subject.
[0502] In some other embodiments, the neoantigen sequence is a
universal neoantigen sequence. In some embodiments, the neoantigen
sequence is a universal neoantigen vaccine. In some embodiments,
the neoantigen sequence is capable of binding to at least one HLA
allele expressed in at least 10%, at least 15%, at least 20%, at
least 25%, at least 30%, at least 35%, at least 40%, or at least
45% of subjects in a population of subjects suffering from the
neoplastic disorder. In some embodiments, the neoantigen sequence
is capable of eliciting a T-cell response against a tumor present
in at least 1%, at least 5%, or at least 10% of a population of
subjects suffering from the neoplastic disorder.
[0503] In some embodiments, the neoantigen sequence has been
identified by sequencing at least one neoantigen mRNA induced in
the subject by administering a therapeutically effective amount of
the at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing. In some
embodiments, the at least one neoantigen mRNA encodes a neoantigen
sequence induced by contacting a neoplastic cell with a
therapeutically effective amount of the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing. In some embodiments, the neoplastic cell is
present in an in vitro cell culture. In some embodiments, the
neoplastic cell is obtained from the subject. In some embodiments,
the neoplastic cell is present in the subject.
[0504] In some embodiments, the neoantigen vaccine comprises at
least one neoantigen mRNA and a pharmaceutically acceptable
carrier. In some embodiments, the at least one neoantigen mRNA is
linked to the pharmaceutically acceptable carrier. In some
embodiments, the pharmaceutically acceptable carrier is chosen from
a peptide, a serum albumin, a keyhole limpet hemocyanin, an
immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an
attenuated toxoid derivative, a cytokine, and a chemokine.
[0505] In some embodiments, the neoantigen vaccine comprises at
least one neoantigen mRNA and a pharmaceutically acceptable
diluent. In some embodiments, the neoantigen vaccine comprises at
least one neoantigen mRNA and a pharmaceutically acceptable
adjuvant (e.g., an adjuvant as described herein).
[0506] In some embodiments, the neoantigen mRNA is encapsulated by
an encapsulating agent. In some embodiments, the encapsulating
agent protects the neoantigen mRNA from degradation and improves
vaccine delivery (McNamara et al. (2015) J Immunol Res.
2015:794528). In some embodiments, the encapsulating agent is a
liposome. In some embodiments, the liposome is a cationic liposome
such as N-[1-(2,3-dioleoloxy)propyl]-N,N,N-trimethyl ammonium
chloride 1 (DOTAP). In some embodiments, the encapsulating agent is
a nanoparticle. In some embodiments, the nanoparticle protects the
neoantigen mRNA from nuclease degradation and/or enhances cell
uptake and/or delivery efficiency. In some embodiments, the
nanoparticle may be engineered to be fully degradable. In some
embodiments, the nanoparticle is a biodegradable core-shell
structured nanoparticle with a pH responsive poly-(b-amino ester)
(PBAE) core enveloped by a phospholipid shell (Su et al. (2011) Mol
Pharm. 8(3):774-87). In some embodiments, such nanoparticles are
particularly efficient in delivering mRNA in vivo and eliciting an
anti-tumor immune response.
[0507] In some embodiments, the subject has a non-synonymous
mutational burden of about 150 mutations or less. In some
embodiments, the subject has a non-synonymous mutational burden of
about 100 mutations or less. In some embodiments, the subject has a
non-synonymous mutational burden of about 50 mutations or less. In
some embodiments, the subject has or is suspected of having a
neoplastic disorder, e.g., a hematological malignancy or a solid
tumor. In some embodiments, the hematological malignancy is chosen
from a B-cell malignancy, a leukemia, a lymphoma, and a myeloma. In
some embodiments, the hematological malignancy is chosen from acute
myeloid leukemia and multiple myeloma. In some embodiments, the
solid tumor is chosen from breast cancer, gastric cancer, prostate
cancer, ovarian cancer, lung cancer, uterine cancer, salivary duct
carcinoma, melanoma, colon cancer, and esophageal cancer. In some
embodiments, the solid tumor is chosen from HER2-positive breast
cancer, gastric adenocarcinoma, and prostate cancer.
[0508] As used herein, "adjuvant" refers to a substance that is
capable of increasing, amplifying, or modulating an immune response
to an accompanying immunogenic agent, e.g., a neoantigen peptide or
mRNA. In certain embodiments, a neoantigen of the present
disclosure can be administered in combination with adjuvants, i.e.,
substances that do not themselves cause adaptive immune responses,
but amplify or modulate the response to an accompanying neoantigen.
A variety of adjuvants can be used in combination with the
disclosed neoantigens, in order to elicit an immune response. In
some embodiments, the adjuvant(s) are chosen to augment the
intrinsic response to the neoantigen without causing conformational
changes in the neoantigen that would affect the qualitative form of
the response. In some embodiments, the adjuvant(s) are chosen to
enhance T-effector (e.g., CD8) cell priming and/or activation.
[0509] In certain embodiments, the adjuvant is an aluminum salt
(alum), such as aluminum hydroxide, aluminum phosphate, and
aluminum sulphate. Such adjuvants can be used with or without other
specific immunostimulating agents, such as 3 de-O-acylated
monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino
acids, such as polyglutamic acid or polylysine. Such adjuvants can
be used with or without other specific immunostimulating agents,
such as muramyl peptides (e.g.,
N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP),
N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),
N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-
-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE),
N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy
propylamide (DTP-DPP)), or other bacterial cell wall components.
Other adjuvants are oil-in-water emulsions and include (a) MF59 (WO
90/14837), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span 85
(optionally containing various amounts of MTP-PE) formulated into
submicron particles using a microfluidizer such as Model 110Y
microfluidizer (Microfluidics), (b) SAF, containing 10% Squalene,
0.4% Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP,
either microfluidized into a submicron emulsion or vortexed to
generate a larger particle size emulsion, and (c) Ribi.TM. adjuvant
system (RAS), (Ribi ImmunoChem) containing 2% squalene, 0.2% Tween
80, and one or more bacterial cell wall components from the group
consisting of monophosphoryllipid A (MPL), trehalose dimycolate
(TDM), and cell wall skeleton (CWS), for example MPL-FCWS
(Detox.TM.). In some embodiments, the adjuvant is a saponin, such
as Stimulon.TM. (QS21) or particles generated therefrom such as
ISCOMs (immunostimulating complexes) and ISCOMATRIX. Other
adjuvants include Complete Freund's Adjuvant (CFA) and Incomplete
Freund's Adjuvant (IFA), cytokines, such as interleukins (IL-1,
IL-2, and IL-12), macrophage colony stimulating factor (M-CSF), and
tumor necrosis factor (TNF).
[0510] An adjuvant can be administered with an immunogenic agent
(e.g., a neoantigen peptide or mRNA) as a single composition, or
can be administered before, concurrent with, or after
administration of the immunogenic agent. In some embodiments, the
immunogenic agent and adjuvant can be packaged and supplied in the
same vial or can be packaged in separate vials and mixed before
use. In some embodiments, the immunogenic agent and adjuvant can be
packaged with a label, indicating the intended therapeutic
application. In some embodiments, if the immunogenic agent and
adjuvant are packaged separately, the packaging can include
instructions for mixing before use. The choice of an adjuvant
and/or carrier depends on the stability of the immunogenic
formulation containing the adjuvant, the route of administration,
the dosing schedule, the efficacy of the adjuvant for the species
being vaccinated, and, in humans, a pharmaceutically acceptable
adjuvant is one that has been approved or is approvable for human
administration by pertinent regulatory bodies. For example,
Complete Freund's adjuvant is not suitable for human
administration. However, alum, MPL or Incomplete Freund's adjuvant
(Chang et al. (1998) Adv Drug Deliv Rev. 32:173-186) alone or
optionally in combination with any of alum, QS21, and MPL and all
combinations thereof are suitable for human administration.
[0511] In some embodiments, the present disclosure further provides
methods of screening for and identifying at least one neoantigen.
More specifically, in some embodiments, the present disclosure
provides a method of identifying at least one neoantigen by (a)
contacting a neoplastic cell with a therapeutically effective
amount of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing; (b)
detecting at least one alternatively-spliced mRNA transcript after
contacting the neoplastic cell with the at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing; (c) predicting translation of the at least
one alternatively-spliced mRNA transcript into at least one
peptide; and (d) comparing the at least one peptide to a reference
proteome, wherein at least one neoantigen is identified if the at
least one peptide does not match any peptides in the reference
proteome. In some embodiments, the method further comprises
contacting one or more additional neoplastic cells to identify at
least one universal neoantigen. In some embodiments, the method is
repeated on one or more additional neoplastic cells or samples
(e.g., a tissue biopsy) to confirm suitable neoantigens (e.g., for
use in a neoantigen vaccine) and/or to identify one or more
universal neoantigens.
[0512] In various other embodiments, the present disclosure
provides a method of identifying at least one neoantigen by (a)
contacting a neoplastic cell with a therapeutically effective
amount of at least one compound chosen from compounds of Formula I,
compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing; (b)
detecting at least one peptide comprising a potential neoantigen
sequence after contacting the neoplastic cell with the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing; and (c) comparing the at least one peptide
to a reference proteome, wherein at least one neoantigen is
identified if the at least one peptide does not match any peptides
in the reference proteome. In some embodiments, the method further
comprises contacting one or more additional neoplastic cells to
identify at least one universal neoantigen. In some embodiments,
the method is repeated on one or more additional neoplastic cells
or samples (e.g., a tissue biopsy) to confirm suitable neoantigens
(e.g., for use in a neoantigen vaccine) and/or to identify one or
more universal neoantigens.
[0513] In some embodiments of the neoantigen identification methods
described herein, detecting at least one alternatively-spliced mRNA
transcript comprises RNAseq. In some embodiments, predicting
translation of the at least one alternatively-spliced mRNA
transcript comprises quantifying the change in percent spliced in
(dPSI) value for the at least one transcript. In some embodiments,
predicting translation of the at least one alternatively-spliced
mRNA transcript comprises RiboSeq and/or ribosomal profiling.
[0514] In some embodiments of the neoantigen identification methods
described herein, the methods further comprise evaluating the at
least one peptide for predicted major histocompatibility complex
(MHC) binding. In some embodiments, predicted MHC binding is
determined by measuring raw affinity predicted binding strength of
the at least one peptide. In some embodiments, a raw affinity
predicted binding strength of about 500 nM or higher indicates MHC
binding. In some embodiments, predicted MHC binding is determined
by identifying a distribution of predicted binding strengths for a
series of random peptides; and comparing predicted binding strength
of the at least one peptide to the distribution. In some
embodiments, a predicted binding strength in the top 2% of the
distribution indicates weak MHC binding. In some embodiments, a
predicted binding strength in the top 0.5% of the distribution
indicates strong MHC binding.
[0515] In some embodiments of the neoantigen identification methods
described herein, the neoplastic cell is present in an in vitro
cell culture. In some embodiments, the neoplastic cell is obtained
from the subject. In some embodiments, the neoplastic cell is
present in the subject.
[0516] Also provided herein, in some embodiments, are methods of
making a neoantigen vaccine by (a) identifying at least one
neoantigen (e.g., at least one neoantigen peptide or its encoding
mRNA) using any of the exemplary identification methods disclosed
herein; and (b) formulating the at least one neoantigen together
with a pharmaceutically acceptable carrier, diluent, or adjuvant
(e.g., any of the pharmaceutically acceptable carriers, diluents,
or adjuvants described herein).
[0517] In some embodiments, the at least one neoantigen and/or
antigenic portion ranges from about 10 to about 50 amino acids in
length. In some embodiments, the at least one neoantigen peptide
ranges from about 10 to about 35 amino acids in length. In some
embodiments, the at least one neoantigen and/or antigenic portion
ranges from about 15 to about 25 amino acids in length. In some
embodiments, the at least one neoantigen and/or antigenic portion
ranges from about 10 to about 20 amino acids in length. In some
embodiments, the at least one neoantigen and/or antigenic portion
does not exclusively overlap or consist of the canonical peptide
sequence (e.g., any of the exemplary canonical peptide sequences
underlined in Table 13).
[0518] In some embodiments, the at least one neoantigen used in the
vaccine is linked to the pharmaceutically acceptable carrier. In
some embodiments, the pharmaceutically acceptable carrier is chosen
from a peptide, a serum albumin, a keyhole limpet hemocyanin, an
immunoglobulin, a thyroglobulin, an ovalbumin, a toxoid or an
attenuated toxoid derivative, a cytokine, and a chemokine.
[0519] In some embodiments, a patient having a cancer as described
herein can be treated with a combination of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing and one or more engineered tumor-targeting
T-cells (i.e., CAR-T). Thus, in some embodiments, the present
disclosure provides a method of treating a subject having or
suspected of having a neoplastic disorder by administering to the
subject a therapeutically effective amount of at least one compound
chosen from compounds of Formula I, compounds of Formula II,
compounds of Formula III, and pharmaceutically acceptable salts of
any of the foregoing; and engineered tumor-targeting T-cells (i.e.,
CAR-T). In some embodiments, a chimeric T-cell receptor can be
engineered using antigen recognition sequences that are reactive
with an identified neoantigen.
[0520] For instance, in some embodiments, in order to target
changes in the extracellular domains of cell surface proteins
induced by at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing, a
chimeric antigen-reactive T-cell receptor (CAR) may be engineered
by first identifying antibodies that recognize a cell
surface-expressed neoantigen protein domain. The antigen
recognition sequences of such antibodies can then be fused to a
T-cell receptor domain for selective targeting and activation.
[0521] In various other embodiments, a strategy integrating the
antigen presentation machinery of tumor cells together with
neoantigens derived from at least one compound chosen from
compounds of Formula I, compounds of Formula II, compounds of
Formula III, and pharmaceutically acceptable salts of any of the
foregoing is employed. In some embodiments, cells containing known
and frequently represented HLA alleles (e.g., HLA-A*02:01) can be
treated with at least one compound chosen from compounds of Formula
I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing and
MHC1-bound neoantigens are identified by ligandomics. In some
embodiments, these peptides can be used to prime and/or expand
T-cells from healthy donors expressing the same HLA allele. Such
T-cells, in some embodiments, can be isolated and the T-cell
receptor (TCR) .alpha. and .beta. chains sequenced to identify the
cognate antigen recognition/variable regions. In some embodiments,
a cognate CAR can then be engineered.
[0522] In some embodiments, the CAR sequences are cloned into
patient-derived T-cell populations and expanded using currently
available protocols. In some embodiments, the engineered T-cells
are then transfused back into the patient's circulation, before,
simultaneously with, or following treatment with at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing. After treatment with the at least one
compound chosen from compounds of Formula I, compounds of Formula
II, compounds of Formula III, and pharmaceutically acceptable salts
of any of the foregoing, in some embodiments, the tumor cells may
begin to present an antigen, e.g., an antigen targeted by the
engineered T-cell population. In some embodiments, the engineered
T-cell population can engage with and kill antigen presenting tumor
cells.
[0523] In order that the disclosure described herein may be more
fully understood, the following examples are set forth. It should
be understood that these examples are for illustrative purposes
only and are not to be construed as limiting this disclosure in any
manner.
EXAMPLES 1-205
[0524] General: Microwave heating was done using a Biotage Emrys
Liberator or Initiator microwave. Column chromatography was carried
out using an Isco Rf200d. Solvent removal was carried out using
either a BUchi rotary evaporator or a Genevac centrifugal
evaporator. Preparative LC/MS was conducted using a Waters
autopurifier and 19.times.100 mm XTerra 5 micron MS C18 column
under acidic mobile phase condition. NMR spectra were recorded
using a Varian 400 MHz spectrometer.
[0525] When the term "inerted" is used to describe a reactor (e.g.,
a reaction vessel, flask, glass reactor, and the like) it is meant
that the air in the reactor has been replaced with an essentially
moisture-free or dry, inert gas (such as nitrogen, argon, and the
like).
[0526] General methods and experimentals for preparing compounds of
the present disclosure are set forth below. In certain cases, a
particular compound is described by way of example. However, it
will be appreciated that in each case a series of compounds of the
present disclosure were prepared in accordance with the schemes and
experimentals described below.
[0527] The following abbreviations are used herein: [0528] COMU:
(1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbeni-
um hexafluorophosphate [0529] DMAP: 4-(Dimethylamino)pyridine
[0530] DMP: Dess Martin Periodinane [0531] EDC:
N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide [0532] KHMDS:
Potassium bis(trimethylsilyl)amide [0533] LCMS: Liquid
chromatography--mass spectrometry [0534] Pd.sub.2(dba).sub.3:
Tris(dibenzylideneacetone)dipalladium(0) [0535] TBAF:
Tetrabutylammonium fluoride [0536] TBSCl: tert-Butyldimethylsilyl
chloride [0537] TBSOTf: tert-Butyldimethylsilyl
trifluoromethanesulfonate [0538] TESCl: Chlorotriethylsilane [0539]
THF: Tetrahy drofuran [0540] TLC: Thin-layer chromatography [0541]
pTsOH: p-Toluenesulfonic acid [0542] PPTS: Pyridinium
p-toluenesulfonate
[0543] Materials: The following compounds are commercially
available and/or can be prepared in a number of ways well known to
one skilled in the art of organic synthesis. More specifically,
disclosed compounds can be prepared using the reactions and
techniques described herein. In the description of the synthetic
methods described below, it is to be understood that all proposed
reaction conditions, including choice of solvent, reaction
atmosphere, reaction temperature, duration of the experiment, and
workup procedures, can be chosen to be the conditions standard for
that reaction, unless otherwise indicated. It is understood by one
skilled in the art of organic synthesis that the functionality
present on various portions of the molecule should be compatible
with the reagents and reactions proposed. Substituents not
compatible with the reaction conditions will be apparent to one
skilled in the art, and alternate methods are therefore indicated.
The starting materials for the examples are either commercially
available or are readily prepared by standard methods from known
materials.
[0544] LCMS information: Mobile phases: A (0.1% formic acid in
H.sub.2O) and B (0.1% formic acid in acetonitrile). Gradient: B
5%.fwdarw.95% in 1.8 minutes. Column: Acquity BEH C18 column (1.7
um, 2.1.times.50 mm).
[0545] U.S. Pat. Nos. 7,884,128 and 7,816,401, both entitled:
Process for Total Synthesis of Pladienolide B and Pladienolide D,
describe methods known in the art for synthesis of Pladienolide B
and D. Synthesis of Pladienolide B and D may also be performed
using methods known in the art and described in Kanada et al.,
"Total Synthesis of the Potent Antitumor Macrolides Pladienolide B
and D," Angew. Chem. Int. Ed. 46:4350-4355 (2007). Kanada et al.
and PCT application publication WO 2003/099813, entitled: Novel
Physiologically Active Substances, describe methods known in the
art for the synthesis of E7107 (Compound 45 of WO '813) from
Pladienolide D (11107D of WO '813). A corresponding U.S. Pat. No.
is 7,550,503 to Kotake et al.
[0546] Exemplified Synthesis of Compounds [0547] Compounds 1-60
(Table I) were prepared by the method of Scheme 1.
##STR00117##
[0547] General Protocol for the Synthesis of Compounds 1-60:
[0548] Step 1: A solution of pladienolide D (A, 5.3 g, 9.7 mmol,
1.0 equiv.) under nitrogen in DMF (80 mL, 0.1M) at 0.degree. C. was
treated with imidazole (4.6 g, 67.8 mmol, 7.0 equiv.) and TBSCl
(7.3 g, 48.4 mmol, 5.0 equiv.). The reaction was allowed to warm to
room temperature and stirred for 20 hours, or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
extracted with ethyl acetate and the organic layer was washed with
brine, dried over sodium sulfate, filtered, and concentrated in
vacuo. The resulting oil was purified by silica gel column
chromatography (hexanes/ethyl acetate as eluant) to afford the
desired product (B, 7.5 g, 9.6 mmol, 99%).
[0549] Step 2: To a solution of olefin B (7.6 g, 9.7 mmol, 1.0
equiv.) in degassed THF:H.sub.2O (210 mL:21 mL, 0.01M) under
nitrogen at 0.degree. C. was added osmium tetroxide (24.4 mL, 1.9
mmol, 0.2 equiv., 2.5% solution in tent-butanol) followed by
N-methylmorpholine N-oxide (2.3 g, 19.5 mmol, 2.0 equiv.). The
reaction was allowed to warm to room temperature and stirred for 13
hours, or until the reaction was determined to be complete by LCMS
or TLC. The reaction was quenched with sodium sulfite, diluted with
ethyl acetate, and the organic layer was washed with water, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(C, 6.8 g, 8.3 mmol, 86%).
[0550] Step 3: To a solution of diol C (7.9 g, 9.7 mmol, 1.0
equiv.) in benzene (350 mL, 0.03M) under nitrogen at room
temperature was added lead tetraacetate (8.6 g, 19.4 mmol, 2.0
equiv.). The reaction was stirred for 30 minutes, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was concentrated and purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product (D,
2.5 g, 5.26 mmol, 54%).
[0551] Step 4: To a solution of aldehyde D (1.4 g, 2.9 mmol, 1.0
equiv.) in THF (9.5 mL, 0.5M) was added ethoxyethene (11.1 mL, 40.0
equiv.) and pyridinium p-toluenesulfonate (0.07 g, 0.3 mmol, 0.1
equiv.) at room temperature. The reaction was stirred for 24 hours,
or until the reaction was determined to be complete by LCMS or TLC.
The reaction was quenched with sodium bicarbonate and diluted with
ethyl acetate. The ethyl acetate was washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product (E,
1.2 g, 2.2 mmol, 75%).
[0552] Step 5: To a solution of corresponding sulfone (1.5 equiv.)
in THF (0.02M) under nitrogen at -78.degree. C. was added KHMDS
(1.5 equiv.) dropwise and the reaction was stirred for 20 minutes.
Then aldehyde E (1.0 equiv.) in THF was added dropwise. The
reaction was stirred at -78.degree. C. for 90 minutes and then
allowed to warm to -20.degree. C. for 1 hour. The reaction was
quenched with ammonium chloride, diluted with ethyl acetate, and
warmed to room temperature. The organic layer was washed with
water, brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (hexane/ethyl acetate as eluent) to afford
the desired product (F).
[0553] Step 6: To a solution of acetate F (1.0 equiv.) in methanol
(0.1M) at room temperature was added potassium carbonate (1.1
equiv.). The reaction was run for 24 hours, or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
quenched with water, diluted with ethyl acetate, washed with brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil (G) was advanced into the next step without
additional purification.
[0554] Step 7: To a solution of alcohol (G) (1.0 equiv.) in
dichloromethane (0.1M) at room temperature was added
N,N-dimethylaminopyridine (0.5 equiv.) followed by 4-nitrophenyl
chloroformate (2.0 equiv.). The reaction was stirred at room
temperature for three hours. Next, the corresponding amine (3.0
equiv.) was added at room temperature. After stirring for one hour,
the reaction was quenched with water and diluted with
dichloromethane. The organic layer was washed with 1N sodium
hydroxide solution, and the organic layer was concentrated. The
resulting oil was purified by silica gel column chromatography
(hexanes/ethyl acetate as eluant) to afford the desired product
(H).
[0555] Step 8: To a solution of silyl ether (H, 1.0 equiv.) in
methanol (0.1M) at room temperature was added
p-methoxytoluenesulfonic acid (3.0 equiv.). The reaction was
stirred for 3 hours, or until the reaction was determined to be
complete by LCMS or TLC. The reaction was quenched with sodium
bicarbonate, diluted with ethyl acetate, washed with water and
brine, dried over magnesium sulfate, filtered, and concentrated in
vacuo. The resulting oil was purified by silica gel column
chromatography (hexane/ethyl acetate as eluent) to afford the
desired product (1-59).
Exemplified Protocol for the Synthesis of compound 46
[0556] Steps 1-4 as above.
[0557] Step 5: To a solution of
(S)-2-(1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridine
(233.0 mg, 0.7 mmol, 1.4 equiv.) in THF (2.5 mL, 0.2M) under
nitrogen at -78.degree. C. was added KHMDS (1.5 mL, 0.75 mmol, 1.5
equiv.) dropwise and the reaction was stirred for 20 minutes. Then
aldehyde E (280.0 mg, 0.5 mmol, 1.0 equiv.) in THF (0.5 mL) was
added dropwise. The reaction was stirred at -78.degree. C. for 90
minutes and then allowed to warm to -20.degree. C. over 1 hour. The
reaction was quenched with ammonium chloride, diluted with ethyl
acetate, and warmed to room temperature. The organic layer was
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel column chromatography (hexane/ethyl acetate as eluent) to
afford the desired Julia product (F, 180 mg, 0.3 mmol, 54%).
[0558] Step 6: To a solution of acetate F (250.0 mg, 0.4 mmol, 1.0
equiv.) in methanol (3 mL, 0.1M) at room temperature was added
potassium carbonate (58.0 mg, 0.4 mmol, 1.1 equiv.). The reaction
was run for 24 hours, or until the reaction was determined to be
complete by LCMS or TLC. The reaction was quenched with water,
diluted with ethyl acetate, washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The resulting foamy
solid (G, 235 mg, 0.4 mmol, 100%) was advanced into the next step
without additional purification.
[0559] Step 7: To a solution of alcohol G (22.0 mg, 0.04 mmol, 1.0
equiv.) in dichloromethane (0.5 mL, 0.1M) at room temperature was
added N,N-dimethylaminopyridine (2.1 mg, 0.02 mmol, 0.5 equiv.)
followed by 4-nitrophenyl chloroformate (14.4 mg, 0.08 mmol, 2.0
equiv.). The reaction was stirred at room temperature for three
hours. Next, 1-(tetrahydro-2H-pyran-4-yl)piperazine (20.4 mg, 0.12
mmol, 3.0 equiv.) was added at room temperature. After stirring for
one hour, the reaction was quenched with water and diluted with
dichloromethane. The organic layer was washed with 1N sodium
hydroxide solution, and the organic layer was concentrated. The
resulting oil was purified by silica gel column chromatography
(hexanes/ethyl acetate as eluant) to afford the desired product (H,
26.0 mg, 0.03 mmol, 80%).
[0560] Step 8: To a solution of silyl ether (H, 26.0 mg, 0.03 mmol,
1.0 equiv.) in methanol (0.3 mL, 0.1M) at room temperature was
added p-methoxytoluenesulfonic acid (17.0 mg, 0.09 mmol, 3.0
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate, diluted with ethyl acetate,
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel column chromatography (hexane/ethyl acetate as eluent) to
afford the desired product (compound 46, 16.3 mg, 0.025 mmol, 85%).
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:8.48 (ddd, J=4.9, 1.9,
1.0 Hz, 1H), 7.54 (td, J=7.7, 1.9 Hz, 1H), 7.09 (d, J=8.0 Hz, 1H),
7.05 (t, J=6.1 Hz, 1H), 6.15-6.34 (m, 1H), 6.04 (d, J=10.8 Hz, 1H),
5.93 (dd, J=15.1, 7.5 Hz, 1H), 5.48-5.67 (m, 2H), 5.08 (d, J=10.5
Hz, 1H), 4.94 (d, J=9.5 Hz, 1H), 3.95 (dd, J=11.3, 3.8 Hz, 2H),
3.53-3.76 (m, 2H), 3.37-3.49 (m, 5H), 3.22-3.37 (m, 2H), 2.35-2.57
(m, 7H), 1.88 (s, 1H), 1.44-1.70 (m, 11H), 1.14-1.39 (m, 8H),
0.72-0.89 (m, 3H), MS (ES+)=626.6[M+H].
TABLE-US-00004 TABLE 1 Structures and analytical data for Compounds
1-60 LCMS data Structure, Compound #, and Chemical Name .sup.1H NMR
data (ES+) ##STR00118## .sup.1H NMR (400 MHz, METHANOL- d4)
.delta.: 0.91 (d, J = 6.78 Hz, 3 H) 1.07 (d, J = 6.65 Hz, 3 H) 1.23
(s, 3 H) 1.30- 1.43 (m, 4 H) 1.55-1.79 (m, 7H) 1.84-2.01 (m, 4 H)
2.28-2.34 (m, 5 H) 2.42 (t, J = 4.96 Hz, 4 H) 2.51- 2.66 (m, 3 H)
3.41 (td, J = 6.87, 1.94 Hz, 2 H) 3.48 (t, J = 6.78 Hz, 2 H)
3.51-3.86 (m, 4 H) 4.96 (d, J = 9.66 Hz, 1 H) 5.07 (d, J = 10.67
Hz, 1 H) 5.55-5.68 (m, 2 H) 5.74 (dd, J = 15.18 9.66 Hz, 1 H) 6.12
(d, J = 10.79 Hz, 1 H) 6.29 (dt, J = 14.81, 10.54 Hz, 1 H) 604.3 1
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6S)-6-methyl-9-oxo-9-
pyrrolidin-1-ylnona-2,4-dien-2-yl]-12-oxo-1-
oxacyclododec-4-en-6-yl] 4-methylpiperazine-1- carboxylate
##STR00119## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.73-0.91
(m, 5 H) 0.92-1.04 (m, 5 H) 1.07-1.33 (m, 5 H) 1.37- 1.59 (m, 3 H)
1.60-1.71 (m, 2 H) 1.80 (s, 1 H) 1.86-2.07 (m, 2 H) 2.24-2.52 (m, 3
H) 2.89-3.09 (m, 1 H) 3.34-3.50 (m, 1 H) 3.52-3.62 (m, 1H)
3.64-3.75 (m, 1 H) 3.76- 4.03 (m, 2 H) 4.83 (s, 1 H) 4.87- 5.04 (m,
1 H) 5.24-5.53 (m, 2 H) 5.53-5.69 (m, 1 H) 5.99 (d, J = 11.04 Hz, 1
H) 6.24 (dd, J = 15.18, 10.92 Hz, 1 H) 534.3 2
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-
[(2E,4E,6S)-7-[[(2R,3R)-3-hydroxypentan-2-
yl]carbamoyloxy]-6-methylhepta-2,4-dien-2-yl]-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] acetate ##STR00120##
.sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.87-0.97 (m, 3 H)
0.97-1.15 (m, 5 H) 1.21 (s, 1 H) 1.29-1.40 (m, 1 H) 1.46-1.70 (m, 5
H) 1.72-1.77 (m, 1 H) 1.82 (s, 1 H) 2.08 (s, 1 H) 2.18 (s, 1 H)
2.35-2.63 (m, 3 H) 3.05 (t, J = 7.03 Hz, 2 H) 3.14-3.27 (m, 3
H)3.50 (dd, J = 3.26, 1.51 Hz, 1 H) 3.62-3.79 (m, 3 H) 3.79-4.08
(m, 3 H) 4.96 (br. s., 1 H) 5.07 (d, J = 9.79 Hz, 1 H) 5.38-5.52
(m, 1 H) 5.54-5.72 (m, 2 H) 5.97 (dd, J = 17.07, 10.79 Hz, 1 H)
6.21-6.49 (m, 1 H) 8.57 (s, 1 H) 3
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6S)-6-methyl-7-
(propylcarbamoyloxy)hepta-2,4-dien-2-yl]-12-oxo-
1-oxacyclododec-4-en-6-yl] acetate ##STR00121## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.53 Hz, 6 H) 1.01- 1.15
(m, 3 H) 1.21 (s, 3 H) 1.29- 1.47 (m, 2 H) 1.50-1.71 (m, 4 H) 1.77
(d, J = 1.00 Hz, 3 H) 2.04-2.09 (m, 3 H) 2.18 (s, 1 H) 2.39 (s, 1
H) 2.48-2.68 (m, 4 H) 2.89 (s, 3 H) 3.15 (s, 1 H) 3.19-3.29 (m, 2
H) 3.31-3.50 (s, 1 H) 3.69 (s, 1 H) 3.74- 3.88 (m, 1 H) 3.89-4.15
(m, 2 H) 5.07 (d, J = 9.79 Hz, 2 H) 5.51-5.64 (m, 1 H) 5.64-5.76
(m, 2 H) 6.09- 6.19 (m, 1 H) 6.37 (ddd, J = 15.12, 10.85, 0.88 Hz,
1 H) 7.25 (dd, J = 8.28, 1.00 Hz, 1 H) 7.73 (d, J = 8.03 Hz, 1 H)
##STR00122## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.90 (d,
J = 6.78 Hz, 3 H) 1.09 (d, J = 6.78 Hz, 3 H) 1.21 (s, 3 H) 1.34-
1.46 (m, 2 H) 1.54-1.67 (m, 2 H) 1.77 (d, J = 0.88 Hz, 3 H)
1.82-1.95 (m, 4 H) 2.08 (s, 3 H) 2.49-2.70 (m, 4 H) 3.34-3.39 (m, 4
H) 3.75-3.87 (m, 1 H) 3.98 (dd, J = 6.78, 1.38 Hz, 2 H) 5.07 (d, J
= 9.91 Hz, 2 H) 5.59 (dd, J = 15.18 9.79 Hz, 1 H) 5.65- 5.76 (m, 2
H) 6.12 (d, J = 10.79 Hz, 1 H) 6.37 (ddd, J = 15.15, 10.82, 1.00
Hz, 1 H) 522.4 5 [(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
pyrrolidine-1-carboxylate ##STR00123## .sup.1H NMR (400 MHz,
METHANOL- d4) .delta.: 0.60-1.79 (m, 15 H) 2.05- 2.35 (m, 5 H) 2.43
(d, J = 3.26 Hz, 6 H) 2.78 (s, 3 H) 2.86-3.00 (m, 3 H) 3.44-3.62
(m, 4 H) 3.64-3.75 (m, 2 H) 3.76-4.05 (m, 6 H) 4.37-4.56 (m, 3 H)
4.95 (d, J = 10.79 Hz, 1 H) 5.41-5.79 (m, 3H) 6.00 (d, J = 10.79
Hz, 1 H) 6.13-6.39 (m, 1 H) 6
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6S)-6-methyl-7-
[methyl(propyl)carbamoyl]oxyhepta-2,4-dien-2-yl]-
12-oxo-1-oxacyclododec-4-en-6-yl] 4-cycloheptyl-
4-oxidopiperazin-4-ium-1-carboxylate ##STR00124## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.65 Hz, 3 H) 1.09 (d, J =
6.78 Hz, 3 H) 1.21 (s, 3 H) 1.31- 1.47 (m, 2 H) 1.62 (dd, J =
15.25, 8.34 Hz, 2 H) 1.75-1.78 (m, 3 H) 2.08 (s, 3 H) 2.51-2.66 (m,
4 H) 2.91 (s, 6 H) 3.81 (br. s., 1 H) 3.97 (dd, J = 6.65, 1.76 Hz,
2 H) 45.07 (d, J = 9.91 Hz, 2 H) 5.51-5.75 (m, 3 H) 6.12 (d, J =
10.92 Hz, 1 H) 6.37 (ddd, J = 15.18, 10.79, 1.00 Hz, 1 H) 518.4 7
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-
(dimethylcarbamoyloxy)-6-methylhepta-2,4-dien-2-
yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1- oxacyclododec-4-en-6-yl]
acetate ##STR00125## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.:
0.89 (d, J = 6.78 Hz, 3 H) 1.06- 1.15 (m, 9 H) 1.21 (s, 3 H) 1.28-
1.45 (m, 2 H) 1.54-1.70 (m, 2 H) 1.74-1.78 (m, 3 H) 2.08 (s, 3 H)
2.51-2.68 (m, 4 H) 3.76-3.87 (m, 546.4 1 H) 3.93-4.03 (m, 2 H) 5.06
(d, J = 9.79 Hz, 2 H) 5.55-5.75 (m, 3 H) 6.12 (d, J = 10.79 Hz, 1
H) 6.37 (ddd, J = 15.12, 10.85, 0.88 Hz, 1 H) 8
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-
(diethylcarbamoyloxy)-6-methylhepta-2,4-dien-2-
yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1- oxacyclododec-4-en-6-yl]
acetate ##STR00126## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.:
0.89 (d, J = 6.65 Hz, 3 H) 1.08 (d, J = 6.78 Hz, 3 H) 1.13 (d, J =
6.78 Hz, 6 H) 1.21 (s, 3 H) 1.29-1.45 (m, 2 H) 1.54-1.71 (m, 2 H)
1.75-1.78 (m, 3 H) 2.08 (s, 3 H) 2.51-2.67 (m, 4 H) 2.76 (s, 3 H)
3.77-3.83 (m, 1 H) 3.98 (d, J = 7.03 Hz, 2 H) 4.33 (br. s., 1 H)
5.06 (d, J = 9.91 Hz, 2 H) 5.55- 5.63 (m, 1 H) 5.65-5.75 (m, 2 H)
6.12 (d, J = 10.92 Hz, 1 H) 6.37 (ddd, J = 15.12, 10.85, 0.88 Hz, 1
H) 546.5 9 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6S)-6-methyl-7-
[methyl(propan-2-yl)carbamoyl]oxyhepta-2,4-dien-
2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl] acetate ##STR00127##
.sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.87-0.98 (m, 6 H)
1.09 (d, J = 6.90 Hz, 3 H) 1.16-1.22 (m, 3 H) 1.32 (br. s., 2 H)
1.38 (d, J = 9.91 Hz, 2 H) 1.49-1.68 (m, 4 H) 1.75-1.78 (m, 3 H)
2.08 (s, 3 H) 2.52-2.66 (m, 4 H) 2.89 (s, 3 H) 3.28-3.37 (m, 18 H)
3.80 (d, J = 6.15 Hz, 1 H) 3.98 (d, J = 7.03 Hz, 2 H) 4.87 (s, 72
H) 5.05- 5.09 (m, 2 H) 5.55-5.62 (m, 1 H) 5.66-5.75 (m, 2 H) 6.12
(d, J = 10.79 Hz, 1 H) 6.33-6.40 (m, 1 H) 560.5 10
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-
[butyl(methyl)carbamoyl]oxy-6-methylhepta-2,4-
dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-6-yl] acetate ##STR00128## .sup.1H NMR (400 MHz,
METHANOL- d4) .delta.: 0.77-0.98 (m, 9 H) 1.01- 1.18 (m, 7 H)
1.20-1.34 (m, 10 H) 1.34-1.53 (m, 5 H) 1.58-1.66 (m, 2 H) 1.76 (s,
3 H) 2.06-2.09 (m, 3 H) 2.50-2.68 (m, 5 H) 2.73 (s, 3 H) 3.94-4.07
(m, 3 H) 5.06 (d, J = 9.79 Hz, 2 H) 5.53-5.64 (m, 1 H) 5.65- 5.75
(m, 2 H) 6.12 (d, J = 10.04 Hz, 1 H) 6.37 (dd, J = 15.18, 10.79 Hz,
1 H) 560.5 11 [(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6S)-7-[butan-2-
yl(methyl)carbamoyl]oxy-6-methylhepta-2,4-dien-
2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-6-yl] acetate ##STR00129## .sup.1H NMR (400 MHz,
METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz, 3 H) 1.08 (d, J = 6.78
Hz, 3 H) 1.21 (s, 3 H) 1.23- 1.30 (m, 2 H) 1.31 (s, 4 H) 1.34- 1.49
(m, 3 H) 1.62 (dd, J = 15.62, 8.22 Hz, 2 H) 1.75-1.79 (m, 2 H)
2.03- 2.09 (m, 3 H) 2.51-2.65 (m, 3 H) 3.15 (s, 1 H) 3.81 (d, J =
3.76 Hz, 1 H) 3.87-3.98 (m, 1 H) 4.59 (s, 4 H) 4.97 (s, 1 H) 5.07
(d, J = 9.79 Hz, 2 H) 5.57 (dd, J = 15.18, 9.79 Hz, 1 H) 5.67-5.77
(m, 2 H) 6.11 (d, J = 10.79 Hz, 1 H) 6.37 (ddd, J = 15.15, 10.82,
1.00 Hz, 1 H) 490.3 12 [(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-
carbamoyloxy-6-methylhepta-2,4-dien-2-yl]-7,10-
dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4- en-6-yl] acetate
##STR00130## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.78 (d,
J = 6.65 Hz, 3 H) 0.80- 0.91 (m, 1 H) 0.95 (d, J = 6.78 Hz, 3 H)
1.09 (s, 3 H) 1.17-1.30 (m, 2 H) 1.40-1.59 (m, 2 H) 1.63-1.73 (m, 4
H) 1.78-1.87 (m, 3 H) 1.96 (s, 3 H) 2.38-2.55 (m, 4 H) 3.19-3.27
(m, 10 H) 3.28-3.41 (m, 1 H) 3.64- 3.75 (m, 1 H) 3.79-3.90 (m, 2 H)
4.46 (s, 1 H) 4.74 (s, 25 H) 4.94 (d, J = 9.66 Hz, 2 H) 5.46 (dd, J
= 15.18, 9.79 Hz, 1 H) 5.54-5.64 (m, 2 H) 6.01 (d, J = 10.92 Hz, 1
H) 6.19-6.31 (m, 1 H) 566.5 13
[(2S,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
(2R)-2-(methoxymethyl)pyrrolidine-1- carboxylate ##STR00131##
.sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz,
3 H) 1.09 (d, J = 6.90 Hz, 3 H) 1.21 (s, 2 H) 1.48- 1.71 (m, 2 H)
1.77 (s, 3 H) 2.08 (s, 3 H) 2.48-2.70 (m, 4 H) 2.95 (s, 3 H)
3.38-3.58 (m, 4 H) 3.69-4.06 (m, 3 H) 5.01-5.16 (m, 2 H) 5.59 (dd,
J = 15.43, 9.79 Hz, 1 H) 5.69 (s, 2 H) 6.12 (d, J = 10.29 Hz, 1 H)
6.38 (dd, J = 15.43, 11.04 Hz, 1 H) 562.3 14
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2- [(2E,4E,6S)-7-[2-
methoxyethyl(methyl)carbamoyl]oxy-6-
methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-6-yl] acetate ##STR00132## .sup.1H NMR (400 MHz,
METHANOL- d4) .delta.: 0.91 (d, J = 6.02 Hz, 3 H) 1.07 (d, J = 6.78
Hz, 3 H) 1.21 (s, 3 H) 1.34- 1.47 (m, 2 H) 1.48-1.49 (m, 1 H)
1.51-1.73 (m, 2 H) 1.78 (d, J = 1.00 Hz, 3 H) 2.08 (s, 3 H)
2.19-2.34 (m, 2 H) 2.46-2.66 (m, 3 H) 3.37 (s, 3 H) 3.74-4.07 (m, 6
H) 5.07 (d, J = 9.79 Hz, 2 H) 5.49-5.80 (m, 3 H) 6.12 (d, J = 10.67
Hz, 1 H) 6.36 (ddd, J = 15.06, 10.67, 1.25 Hz, 1 H) 15
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
azetidine-1-carboxylate ##STR00133## .sup.1H NMR (400 MHz,
METHANOL- d4) .delta.: 0.89 (d, J = 6.78 Hz, 3 H) 1.09 (d, J = 6.78
Hz, 3 H) 1.18 (d, J = 6.27 Hz, 3 H) 1.21 (s, 3 H) 1.28-1.48 (m, 3
H) 1.53-1.69 (m, 3 H) 1.76 (d, J = 1.00 Hz, 3 H) 1.79-2.06 (m, 3 H)
2.08 (s, 3 H) 2.55 (br. s., 4 H) 3.36- 3.45 (m, 1 H) 3.73-4.11 (m,
4 H) 5.06 (m, J = 9.54 Hz, 2 H) 5.58 (dd, J = 15.31, 9.79 Hz, 1 H)
5.65-5.77 (m, 2 H) 6.13 (d, J = 9.79 Hz, 1 H) 6.37 (dd, J = 15.81,
10.54 Hz, 1 H) 536.4 16
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
(2S)-2-methylpyrrolidine-1-carboxylate ##STR00134## .sup.1H NMR
(400 MHz, METHANOL- d4) .delta.: 0.77 (d, J = 6.65 Hz, 3 H) 0.97
(d, J = 6.90 Hz, 3 H) 1.01-1.14 (m, 6 H) 1.19-1.32 (m, 2 H)
1.39-1.57 (m, 3 H) 1.64 (s, 3 H) 1.96 (s, 3 H) 2.36-2.61 (m, 4 H)
3.24-3.34 (m, 2 H) 3.60-3.74 (m, 1 H) 3.74-3.94 (m, 3 H) 4.86-5.00
(m, 2 H) 5.46 (dd, J = 15.18, 9.79 Hz, 1 H) 5.56 (m, 2 H) 5.98 (d,
J = 9.91 Hz, 1 H) 6.25 (dd, J = 16.06, 10.54 Hz, 1 H) 536.3 17
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
(2S)-2-methylpyrrolidine-1-carboxylate ##STR00135## .sup.1H NMR
(400 MHz, METHANOL- d4) .delta.: 0.00-0.00 (m, 1 H) 0.90 (d, J =
6.78 Hz, 3 H) 1.08 (d, J = 6.78 Hz, 3 H) 1.21 (s, 3 H) 1.29-1.70
(m, 12 H) 1.77 (d, J = 1.00 Hz, 3 H) 2.08 (s, 3 H) 2.46-2.71 (m, 4
H) 3.37-3.47 (m, 4 H) 3.73-3.86 (m, 1 H) 3.92- 4.03 (m, 2 H) 5.05
(m, 2 H) 5.59 (dd, J = 15.18, 9.79 Hz, 1 H) 5.64-5.77 (m, 2 H) 6.12
(dd, J = 10.79, 1.00 Hz, 1 H) 6.37 (ddd, J = 15.12, 10.85, 0.88 Hz,
1 H) 18 [(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
piperidine-1-carboxylate
##STR00136## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.90 (d,
J = 6.40 Hz, 3 H) 1.09 (d, J = 6.78 Hz, 3 H) 1.21 (s, 3 H) 1.32-
1.47 (m, 2 H) 1.53-1.69 (m, 2 H) 1.77 (s, 3 H) 1.80-1.89 (m, 1 H)
1.90-2.01 (m, 3 H) 2.08 (s, 3 H) 2.55 (br. s., 4 H) 3.35-3.47 (m, 2
H) 3.47-3.67 (m, 2 H) 3.74-3.90 (m, 2 H) 3.90-4.07 (m, 2 H)
5.02-5.16 (m, 2 H) 5.59 (dd, J = 15.18, 9.79 Hz, 1 H) 5.70 (d, J =
9.66 Hz, 2 H) 6.14 (d, J = 10.67 Hz, 1 H) 6.38 (dd, J = 15.06,
11.04 Hz, 1 H) 19 [(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
(2R)-2-(hydroxymethyl)pyrrolidine-1- carboxylate ##STR00137##
.sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz,
3 H) 1.10 (d, J = 6.78 Hz, 3 H) 1.21 (s, 3 H) 1.33- 1.46 (m, 2 H)
1.53-1.70 (m, 2 H) 1.77 (s, 3 H) 1.85-2.06 (m, 3 H) 2.08 (s, 3 H)
2.47-2.70 (m, 4 H) 3.47 (d, J = 4.39 Hz, 3 H) 3.76-3.86 (m, 1 H)
3.92-4.05 (m, 2 H) 4.31- 4.46 (m, 1 H) 5.01-5.10 (m, 2 H) 5.57 (dd,
J = 15.43 9.79 Hz, 1 H) 5.66-5.82 (m, 2 H) 6.12 (d, J = 10.92 Hz, 1
H) 6.38 (ddd, J = 15.18, 10.85, 0.69 Hz, 1 H) 20
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
(3R)-3-hydroxypyrrolidine-1-carboxylate ##STR00138## .sup.1H NMR
(400 MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.65 Hz, 3 H) 1.08
(d, J = 6.78 Hz, 3 H) 1.21 (s, 3 H) 1.30- 1.45 (m, 2 H) 1.56-1.69
(m, 2 H) 1.74-1.82 (m, 3 H) 2.08 (s, 3 H) 2.47-2.71 (m, 4 H)
3.39-3.55 (m, 4 H) 3.55-3.71 (m, 4 H) 3.73-3.85 (m, 1 H) 3.95-4.06
(m, 2 H) 4.98- 5.16 (m, 2 H) 5.58 (dd, J = 14.93, 9.54 Hz, 1 H)
5.65-5.76 (m, 2 H) 6.13 (d, J = 11.04 Hz, 1 H) 6.38 (ddd, J =
15.18, 10.92, 0.88 Hz, 1 H) 560.1 21
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
morpholine-4-carboxylate ##STR00139## .sup.1H NMR (400 MHz,
METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz, 3 H) 1.08 (d, J = 6.90
Hz, 3 H) 1.21 (s, 3 H) 1.29- 1.45 (m, 3 H) 1.54-1.68 (m, 2 H) 1.77
(d, J = 1.00 Hz, 3 H) 2.08 (s, 3 H) 2.32 (s, 3 H) 2.37-2.47 (m, 4
H) 2.48-2.71 (m, 4 H) 3.44-3.57 (m, 4 H) 3.73-3.88 (m, 1 H)
3.91-4.06 (m, 2 H) 5.02-5.15 (m, 2 H) 5.59 (dd, J = 15.31, 10.04
Hz, 1 H) 5.64- 5.77 (m, 2 H) 6.12 (d, J = 10.92 Hz, 1 H) 6.37 (ddd,
J = 15.15, 10.89, 0.94 Hz, 1 H) 551.2 22
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-acetyloxy-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-2-yl]-2-methylhepta-3,5- dienyl]
4-methylpiperazine-1-carboxylate ##STR00140## .sup.1H NMR (400 MHz,
METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz, 3 H) 1.09 (d, J = 6.78
Hz, 3 H) 1.21 (s, 3 H) 1.31 (s, 4 H) 1.49-1.70 (m, 3 H) 1.73- 1.82
(m, 3 H) 2.08 (s, 3 H) 2.55 (s, 4 H) 3.03 (t, J = 6.34 Hz, 2 H)
3.69 (t, J = 6.27 Hz, 2 H) 3.75-3.86 (m, 1 H) 4.02 (d, J = 6.78 Hz,
2 H) 4.45 (s, 2 H) 4.61-4.61 (m, 1 H) 5.08 (s, 2 H) 5.58 (dd, J =
15.18, 9.79 Hz, 1 H) 5.64-5.79 (m, 2 H) 6.12 (d, J = 10.79 Hz, 1 H)
6.37 (ddd, J = 15.15, 10.82, 1.00 Hz, 1 H) 562.6 23
3-thiazolidinecarboxylic acid [(2R,3E,5E)-6-
[(2R,3S,4E,6R,7R,10R)-6-acetyloxy-7,10-
dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
en-2-yl]-2-methylhepta-3,5-dienyl] ester ##STR00141## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.73-0.92 (m, 4 H) 0.94-1.05 (m, 3
H) 1.08- 1.34 (m, 8 H) 1.38-1.58 (m, 3 H) 1.61 (br. s., 1 H) 1.66
(s, 3 H) 1.71- 1.82 (m, 4 H) 1.87 (d, J = 15.06 Hz, 1 H) 1.97 (s, 1
H) 2.24 (s, 3 H) 2.32 (br. s., 4 H) 2.36-2.58 (m, 4 H) 3.09- 3.38
(m, 4 H)3.39-3.48 (m, 7 H) 3.55-3.73 (m, 2 H) 3.79 (s, 1 H) 3.89
(qd, J = 10.46, 6.78 Hz, 2 H) 4.05 (q, J = 7.03 Hz, 1 H) 4.95 (d, J
= 9.29 Hz, 1 H) 5.09 (d, J = 10.54 Hz, 1 H) 5.23 (s, 1 H) 5.49-5.71
(m, 3 H) 6.02 (d, J = 10.54 Hz, 1 H) 6.20 (dd, J = 15.06, 10.79 Hz,
1 H) ##STR00142## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 6.20
(dd, J = 15.1, 10.8 Hz, 1H), 6.02 (d, J = 10.8 Hz, 1H), 5.37-5.76
(m, 3H), 5.08 (d, J = 10.5 Hz, 1H), 4.95 (d, J = 9.5 Hz, 1H), 4.40
(br. s., 1H), 3.81-4.00 (m, 2H), 3.64-3.73 (m, 1H), 3.10-3.56 (m,
9H), 2.38-2.62 (m, 4H), 2.24 (s, 3H), 1.81-1.99 (m, 3H), 1.57-1.77
(m, 5H), 1.39-1.52 (m, 1H), 1.17 (s, 3H), 1.09-1.37 (m, 5H), 1.00
(d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H) 622.7 25
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-
[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
methylpiperazine-1-carboxylate ##STR00143## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 6.21 (dd, J = 15.1, 10.8 Hz, 1H), 6.02 (d, J
= 10.8 Hz, 1H), 5.50-5.70 (m, 3H), 5.23 (s, 1H), 5.08 (d, J = 10.5
Hz, 1H), 4.95 (d, J = 9.3 Hz, 1H), 3.80- 4.02 (m, 3H), 3.48-3.74
(m, 4H), 3.44 (br. s., 6H), 3.24-3.38 (m, 1H), 2.36-2.59 (m, 5H),
2.32 (br. s., 4H), 2.25 (s, 4H), 1.87-2.08 (m, 2H), 1.58-1.82 (m,
7H), 1.41-1.57 (m, 2H), 1.19-1.36 (m, 3H), 1.17 (s, 3H), 1.08 (br.
s., 1H), 1.00 (d, J = 6.8 Hz, 3H), 0.83 (d, J = 6.8 Hz, 3H) 636.5
26 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-
[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-
1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
methylpiperazine-1-carboxylate ##STR00144## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 7.15-7.24 (m, 4H), 6.23 (dd, J = 14.7, 10.4
Hz, 1H), 6.03 (d, J = 11.0 Hz, 1H), 5.49- 5.69 (m, 2H), 5.08 (d, J
= 10.5 Hz, 1H), 4.95 (d, J = 9.3 Hz, 1H), 4.65 (d, J = 16.3 Hz,
3H), 3.89-4.08 (m, 2H), 3.67 (br. s., 1H), 3.35-3.49 (m, 4H),
2.36-2.63 (m, 3H), 2.30 (br. s., 3H), 2.23 (s, 3H), 1.97 (s, 1H),
1.87 (s, 1H), 1.55-1.73 (m, 4H), 1.40-1.55 (m, 2H), 1.09-1.32 (m,
5H), 0.97- 1.09 (m, 3H), 0.81 (d, J = 6.5 Hz, 3H) 654.5 27
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-
dihydroxy-3,7-dimethyl-6-(4-methylpiperazine-1-
carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-
2-methylhepta-3,5-dienyl] 1,3-dihydroisoindole-2- carboxylate
##STR00145## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 7.63 (d,
J = 8.0 Hz, 1H), 7.25-7.53 (m, 2H), 6.12-6.36 (m, 1H), 6.03 (s,
1H), 6.00 (s, 1H), 5.41-5.67 (m, 2H), 5.04-5.16 (m, 1H), 4.75-5.03
(m, 1H), 4.35- 4.54 (m, 1H), 3.89 (d, J = 6.5 Hz, 1H), 3.60-3.77
(m, 1H), 3.54 (d, J = 11.0 Hz, 1H), 3.44 (br. s., 1H), 3.38 (br.
s., 1H), 3.05-3.32 (m, 1H), 2.69-2.92 (m, 1H), 2.36-2.62 (m, 2H),
2.22- 2.36 (m, 2H), 2.07-2.21 (m, 1H), 1.91-2.06 (m, 1H), 1.87 (d,
J = 10.5 Hz, 1H), 1.55-1.76 (m, 3H), 1.13- 1.41 (m, 4H), 0.94-1.13
(m, 3H), 0.66-0.93 (m, 3H) 652.8 28
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-7,10-
dihydroxy-3,7-dimethyl-6-(4-methylpiperazine-1-
carbonyl)oxy-12-oxo-1-oxacyclododec-4-en-2-yl]-
2-methylhepta-3,5-dienyl] indole-1-carboxylate ##STR00146## .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta.: 0.99-1.12 (m, 3 H) 1.02-1.10
(m, 6 H) 1.16 (d, J = 5.65 Hz, 1 H) 1.24-1.25 (m, 3 H) 1.28-1.42
(m, 2 H) 1.49-1.66 (m, 5 H) 1.74 (d, J = 4.14 Hz, 3 H) 1.92 (m, 1
H) 2.34 (br. s., 3 H) 2.38- 2.48 (m, 4 H) 2.49-2.64 (m, 4 H)
3.23-3.38 (m, 1 H) 3.42-3.62 (m, 6 H) 3.62-3.79 (m, 2 H) 3.98 (d, J
= 6.40 Hz, 2 H) 5.02 (d, J = 9.41 Hz, 1 H) 5.15 (d, J = 10.67 Hz, 1
H) 5.57- 5.73 (m, 3 H) 6.06-6.12 (m, 1 H) 6.28 (dd, J = 16.31,
11.04 Hz, 1 H) 650.5 29 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-
[(2E,4E,6S)-7-[2-(1-hydroxyethyl)pyrrolidine-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
methylpiperazine-1-carboxylate ##STR00147## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.76-1.00 (d, , J = 6.53 Hz 3 H) 1.01-1.12
(d, J = 6.78 Hz, 3 H) 1.23 (s, 3 H) 1.26 (s, 6 H) 1.28-1.43 (m, 4
H) 1.47-1.62 (m, 1 H) 1.72 (s, 3 H) 1.75-1.94 (m, 3 H) 2.51 (s, 3
H) 2.51-2.62 (m, 4 H) 2.74 (br. s., 4 H) 3.41 (m, 1 H) 3.49 (m, 1H)
3.66 (br. s., 4 H) 3.72 (m, 1 H) 3.70-3.82 (m, 2 H) 3.85- 4.05 (m,
2 H) 5.02 (d, J = 9.29 Hz, 1 H) 5.15 (d, J = 10.79 Hz, 1 H) 5.58-
5.73 (m, 3 H) 6.08 (d, J = 10.16 Hz, 1 H) 6.25 (m, 1 H) 634.3 30
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,2-
dimethylpyrrolidine-1-carbonyl)oxy-6-methylhepta-
2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-
1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1- carboxylate
##STR00148## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.90 (d,
J = 6.65 Hz, 3 H) 1.09 (d, J = 6.78 Hz, 3 H) 1.13 (d, J = 6.27 Hz,
3 H) 1.16 (d, J = 6.40 Hz, 3 H) 1.24 (s, 3 H) 1.36-1.46 (m, 2 H)
1.50-1.71 (m, 4 H) 1.76 (s, 3 H) 2.07-2.23 (m, 2 H) 2.51-2.70 (m, 7
H) 2.92 (br. s., 4 H) 3.50-3.90 (m, 5 H) 3.91-4.03 (m, 4 H) 4.97
(d, J = 9.66 Hz, 1 H) 5.07 (d, J = 10.67 Hz, 1 H) 5.61 (dd, J =
15.18, 9.66 Hz, 1 H) 5.65-5.79 (m, 2 H) 6.12 (d, J = 10.79 Hz, 1 H)
6.37 (dd, J = 14.93, 10.79 Hz, 1 H) 634.5 31
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2S,5S)-
2,5-dimethylpyrrolidine-1-carbonyl]oxy-6-
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
methylpiperazine-1-carboxylate ##STR00149## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 7.79 (br. s., 1H), 7.45 (s, 1H), 7.19 (s,
3H), 7.08 (d, J = 7.5 Hz, 2H), 6.77-7.00 (m, 1H), 6.13-6.37 (m,
1H), 6.04 (m, 2H), 5.49-5.70 (m, 3H), 5.23 (s, 1H), 5.02-5.16 (m,
1H), 4.95 (d, J = 9.3 Hz, 1H), 4.09-4.37 (m, 1H), 4.05 (d, J = 6.8
Hz, 1H), 3.83-3.99 (m, 2H), 3.65 (d, J = 19.3 Hz, 1H), 3.34-3.50
(m, 4H), 3.05 (t, J = 8.5 Hz, 2H), 2.64 (br. s., 1H), 2.37-2.60 (m,
3H), 2.30 (br. s., 3H), 2.23 (s, 3H), 1.98 (s, 1H), 1.87 (s, 1H),
1.81 (s, 1H), 1.57- 1.70 (m, 4H), 1.40-1.57 (m, 3H), 1.11-1.31 (m,
5H), 1.05 (d, J = 6.5 Hz, 3H), 0.70-0.92 (m, 3H) 654.5 ##STR00150##
.sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.65 Hz,
3 H) 1.05- 1.14 (m, 3 H) 1.24 (s, 3 H) 1.34- 1.46 (m, 2 H)
1.53-1.71 (m, 2 H) 1.76 (s, 3 H) 1.95-2.30 (m, 2 H) 2.55 (br. s., 5
H) 2.57-2.69 (m, 2 H) 2.74 (br. s., 4 H) 3.40-3.52 (m, 2 H)
3.53-3.86 (m, 7 H) 3.91-4.10 (m, 2 H) 4.97 (d, J = 9.66 Hz, 1 H)
5.07 (d, J = 10.67 Hz, 1 H) 5.25 (dt, J = 52.95, 3.33 Hz, 1 H) 5.60
(dd, J = 15.18, 9.79 Hz, 1 H) 5.65-5.79 (m, 2 H) 6.12 (d, J = 10.79
Hz, 1 H) 6.31-6.43 (m, 1 H) 625.6 33
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-
fluoropyrrolidine-1-carbonyl]oxy-6-methylhepta-
2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-
1-oxacyclododec-4-en-6-yl] 4-methylpiperazine-1- carboxylate
##STR00151## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.91 (d,
J = 6.65 Hz, 3 H) 1.09 (d, J = 6.78 Hz, 3 H) 1.24 (s, 3 H) 1.35-
1.45 (m, 2 H) 1.54-1.71 (m, 2 H) 1.77 (s, 3 H) 1.81-2.10 (m, 4 H)
2.46 (s, 3 H) 2.54 (d, J = 3.64 Hz, 2 H) 2.62 (br. s., 6 H) 2.72
(br. s., 1 H) 3.35-3.47 (m, 3 H) 3.52-3.74 (m, 4 H) 3.78-3.85 (m, 1
H) 4.00 (d, J = 5.14 Hz, 3 H) 4.27-4.54 (m, 2 H) 4.96 (d, J = 9.66
Hz, 1 H) 5.07 (d, J = 10.67 Hz, 1 H) 5.71 (s, 3 H) 6.12 (d, J =
10.16 Hz, 1 H) 6.37 (dd, J = 15.31, 10.92 Hz, 1 H) 639.5 34
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2R)-2-
(fluoromethyl)pyrrolidine-1-carbonyl]oxy-6-
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
methylpiperazine-1-carboxylate ##STR00152## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.89 (d, J = 6.65 Hz, 6 H) 1.08 (d, J = 6.65
Hz, 1 H) 1.14 (br. s., 1 H) 1.23 (s, 3 H) 1.27-1.42 (m, 3 H)
1.45-1.64 (m, 1 H) 1.72 (s, 3 H) 1.73-1.93 (m, 2 H) 1.75-1.93 (m,
15 H) 2.26-2.35 (m, 2 H) 2.35 (s, 3 H) 2.45 (br. s., 4 H) 2.48-2.74
(m, 5 H) 3.39 (m, 1 H) 3.45 (m, 1 H) 3.54 (t, J = 4.89 Hz, 4 H)
3.70-3.78 (m, 1 H) 4.01 (m, 1 H) 4.11 (m, 1 H) 4.36 (d, J = 5.52
Hz, 2 H) 5.02 (d, J = 9.41 Hz, 1 H) 5.15 (d, J = 10.67 Hz, 1 H)
5.57-5.75 (m, 3 H) 6.08 (d, J = 10.67 Hz, 1 H) 6.19- 6.41 (m, 1 H)
648.4 ##STR00153## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.90 (d, J = 6.78 Hz, 3 H) 1.20 (s, 3 H) 1.25- 1.39 (m, 4 H) 1.45
(d, J = 7.03 Hz, 3 H) 1.72 (d, J = 1.00 Hz, 3 H) 2.07 (br. s., 1 H)
2.09 (s, 3 H) 2.45-2.63 (m, 3 H) 3.51 (d, J = 9.41 Hz, 1 H) 3.66-
3.81 (m, 2 H) 5.08 (d, J = 9.16 Hz, 1 H) 5.15 (d, J = 10.54 Hz, 1
H) 5.60- 5.66 (m, 2 H) 5.99 (dd, J = 15.06, 7.40 Hz, 1 H) 6.11 (d,
J = 11.92 Hz, 1 H) 6.27-6.35 (m, 1 H) 7.11 (ddd, J = 7.40, 4.89,
1.13 Hz, 1 H) 7.16 (d, J = 7.91 Hz, 1 H) 7.61 (t, J = 7.36 Hz, 1 H)
8.54 (d, J = 5.02 Hz, 1 H) 472.2 36
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate
##STR00154## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.86 (d,
J = 6.78 Hz, 3 H) 1.20 (s, 3 H) 1.28- 1.53 (m, 4 H) 1.44 (d, J =
7.03 Hz, 3 H) 1.73 (d, J = 1.00 Hz, 3 H) 2.04- 2.12 (m, 4 H)
2.46-2.61 (m, 3 H) 3.52 (d, J = 10.92 Hz, 1 H) 3.66-3.85 (m, 2 H)
5.07 (d, J = 9.03 Hz, 1 H) 5.15 (d, J = 10.67 Hz, 1 H) 5.55-5.71
(m, 2 H) 6.00 (dd, J = 15.12, 7.47 Hz, 1 H) 6.10 (d, J = 10.79 Hz,
1 H) 6.32 (ddd, J = 15.18, 10.79, 1.13 Hz, 1 H) 7.11 (t, J = 6.13
Hz, 1 H) 7.14-7.18 (m, 1 H) 7.26 (s, 2 H) 7.61 (t, J = 7.63 Hz, 1
H) 8.54 (d, J = 5.00 Hz, 1 H) 472.2 37
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate
##STR00155## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.86-0.92
(m, 3 H) 1.21 (s, 3 H) 1.22-1.39 (m, 4 H) 1.33 (d, J = 7.15 Hz, 3
H) 1.42 (m, 3 H) 1.73 (s, 3 H) 2.07-2.11 (m, 4 H) 2.44-2.69 (m, 4
H) 3.03 (br. s., 1 H) 3.48-3.55 (m, 1 H) 3.64-3.83 (m, 3 H)
3.92-3.99 (m, 1 H) 5.08 (d, J = 8.91 Hz, 1 H) 5.15 (d, J = 10.67
Hz, 1 H) 5.63 (t, J = 8.78 Hz, 2 H) 5.94 (dd, J = 15.18, 7.65 Hz, 1
H) 6.10 (d, J = 10.55 Hz, 1 H) 6.24-6.33 (m, 1 H) 6.99-7.04 (m, 2
H) 7.57 (t, J = 7.66 Hz, 1 H) 530.3 38
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-[(2E,4E)-
6-[6-[(2R)-1-hydroxypropan-2-yl]pyridin-2-
yl]hepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-6-yl] acetate ##STR00156## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 8.10-8.34 (m, 1H), 6.93 (s, 1H), 6.38-6.63
(m, 1H), 6.17-6.32 (m, 1H), 5.52-5.73 (m, 1H), 5.30-5.52 (m, 1H),
5.01- 5.16 (m, 1H), 4.95 (d, J = 9.3 Hz, 1H), 3.97-4.09 (m, 1H),
3.93 (br. s., 1H), 3.52 (br. s., 1H), 3.42 (br. s., 1H), 3.35 (br.
s., 1H), 2.74 (t, J = 7.3 Hz, 1H), 2.54 (t, J = 7.4 Hz, 1H), 2.46
(br. s., 1H), 1.92-2.19 (m, 3H), 1.82-1.90 (m, 1H), 1.61-1.81 (m,
2H), 1.50 (br. s., 5H), 1.34-1.45 (m, 2H), 1.01-1.10 (m, 2H), 0.94
(dd, J = 6.7, 4.6 Hz, 1H), 0.64-0.90 (m, 3H) 600.6 39
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E)-6-[2-
(dimethylamino)pyrimidin-4-yl]hepta-2,4-dien-2-
yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1- oxacyclododec-4-en-6-yl]
4-methylpiperazine-1- carboxylate ##STR00157## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta. ppm 0.90 (d, J = 6.78 Hz, 3 H) 1.24 (s,
3 H) 1.28- 1.41 (m, 1 H) 1.45 (d, J = 6.90 Hz, 3 H) 1.51-1.71 (m, 3
H) 1.73 (s, 3 H) 1.91 (s, 1 H), 2.42 (s, 3 H), 2.43- 2.64 (m, 6 H)
3.47 (m, 4 H) 3.58- 3.83 (m, 2 H) 5.01 (d, J = 9.54 Hz, 1 H) 5.14
(d, J = 10.67 Hz, 1 H) 5.51- 5.75 (m, 2 H) 5.99 (dd, J = 15.06,
7.53 Hz, 1 H) 6.11 (d, J = 10.79 Hz, 1 H) 6.27-6.34 (m, 1 H) 7.11
(ddd, J = 7.43, 4.86, 1.13 Hz, 1 H) 7.16 (d, J = 7.78 Hz, 1 H) 7.60
(td, J = 7.69, 1.82 Hz, 1 H) 8.54 (d, J = 5.03 Hz, 1 H) 556.3
##STR00158## .sup.1H NMR (CD.sub.2Cl.sub.2) .delta.: 8.90-9.14 (m,
1H), 7.68 (d, J = 8.0 Hz, 1H), 7.32- 7.54 (m, 1H), 6.29-6.53 (m,
1H), 6.12 (d, J = 10.8 Hz, 1H), 5.85-6.07 (m, 1H), 5.63-5.84 (m,
1H), 5.05- 5.23 (m, 1H), 4.98 (d, J = 9.5 Hz, 1H), 3.70 (br. s.,
1H), 3.48 (br. s., 2H), 3.30 (br. s., 1H), 2.44-2.65 (m, 2H), 2.35
(d, J = 8.8 Hz, 2H), 2.00- 2.20 (m, 1H), 1.82-2.00 (m, 1H),
1.70-1.80 (m, 1H), 1.64 (d, J = 11.0 Hz, 1H), 1.44-1.58 (m, 6H),
1.24- 1.43 (m, 2H), 1.20 (s, 1H), 1.12 (d, J = 5.5 Hz, 1H),
0.66-0.91 (m, 3H) 41 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E)-6-pyridazin-3-ylhepta-
2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] 4-
methylpiperazine-1-carboxylate ##STR00159## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.72-0.88 (m, 4 H) 1.09 (d, J = 6.27 Hz, 2
H) 1.13-1.33 (m, 8 H) 1.36-1.46 (m, 4 H) 1.49 (br. s., 6 H) 1.66
(s, 3 H) 1.82 (br. s., 1 H) 2.00 (d, J = 19.32 Hz, 2 H) 2.30 (br.
s., 3 H) 2.37-2.64 (m, 3 H) 3.07 (d, J = 7.28 Hz, 2 H) 3.29- 3.56
(m, 4 H) 3.67 (br. s., 1 H) 3.73- 3.95 (m, 2 H) 4.95 (d, J = 9.29
Hz, 1 H) 5.08 (d, J = 10.79 Hz, 1 H) 5.23 (s, 1 H) 5.47-5.67 (m, 2
H) 5.93-6.16 (m, 2 H) 6.17-6.41 (m, 1 H) 6.93 (s, 1 H) 6.99-7.13
(m, 1 H) 7.45 (s, 1 H) 8.53-8.74 (m, 2 H) 557.46 42
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E)-6-pyrimidin-2-ylhepta-
2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] 4-
methylpiperazine-1-carboxylate ##STR00160## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 8.57 (d, J = 5.0 Hz, 1H), 7.63 (td, J = 7.7,
1.8 Hz, 1H), 7.18 (d, J = 7.7 Hz, 1H), 7.14 (t, J = 6.1 Hz, 1H),
6.34 (dd, J = 14.6, 10.3 Hz, 1H), 6.14 (d, J = 10.8 Hz, 1H), 6.02
(dd, J = 15.1, 7.5 Hz, 1H), 5.57-5.78 (m, 2H), 5.32 (s, 1H), 5.17
(d, J = 10.8 Hz, 1H), 5.04 (d, J = 9.5 Hz, 1H), 3.60-3.84 (m, 2H),
3.51 (br. s., 4H), 2.76 (br. s., 1H), 2.44-2.67 (m, 6H), 1.98 (s,
1H), 1.66-1.81 (m, 4H), 1.51-1.66 (m, 3H), 1.22-1.49 (m, 8H), 1.08
(d, J = 6.3 Hz, 5H), 0.79-1.00 (m, 3H) 584.5 43
[(2R,3R,4E,6S,7R,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6R)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-propan-2-ylpiperazine-1-carboxylate ##STR00161## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 8.48 (d, J = 4.0 Hz, 1H), 7.54 (td, J =
7.7, 1.9 Hz, 1H), 7.09 (d, J = 7.5 Hz, 1H),7.05 (t, J = 6.2 Hz,
1H), 6.25 (dd, J = 14.6, 11.0 Hz, 1H), 6.04 (d, J = 11.0 Hz, 1H),
5.93 (dd, J = 15.2, 7.4 Hz, 1H), 5.46-5.70 (m, 2H), 5.08 (d, J =
10.5 Hz, 1H), 4.94 (d, J = 9.5 Hz, 1H), 3.54-3.76 (m, 2H),
3.28-3.53 (m, 4H), 2.37-2.59 (m, 5H), 1.89 (br. s., 1H), 1.57-1.71
(m, 4H), 1.42-1.57 (m, 5H), 1.33-1.39 (m, 3H), 1.14- 1.30 (m, 5H),
1.01 (br. s., 6H), 0.77- 0.93 (m, 3H) 596.6 44
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-tert-butylpiperazine-1-carboxylate ##STR00162## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 8.48 (d, J = 4.9 Hz, 1H), 7.54 (td, J =
7.7, 2.0 Hz, 1H), 7.09 (d, J = 8.1 Hz, 1H), 7.05 (t, J = 6.2 Hz,
1H), 6.25 (ddd, J = 15.1, 10.9, 1.1 Hz, 1H), 6.04 (d, J = 10.8 Hz,
1H), 5.93 (dd, J = 15.1, 7.5 Hz, 1H), 5.48-5.67 (m, 2H), 5.08 (d, J
= 10.5 Hz, 1H), 4.94 (d, J = 9.3 Hz, 1H), 3.58-3.73 (m, 2H),
3.36-3.52 (m, 5H), 2.33-2.57 (m, 8H), 1.89 (s, 1H), 1.73-1.83 (m,
2H), 1.57-1.69 (m, 7H), 1.42-1.57 (m, 4H), 1.15- 1.40 (m, 10H),
0.74-0.88 (m, 3H) 610.6 45
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cyclopentylpiperazine-1-carboxylate ##STR00163## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 8.48 (ddd, J = 4.9, 1.9, 1.0 Hz, 1H),
7.54 (td, J = 7.7, 1.9 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 7.05 (t,
J = 6.1 Hz, 1H), 6.15-6.34 (m, 1H), 6.04 (d, J = 10.8 Hz, 1H), 5.93
(dd, J = 15.1, 7.5 Hz, 1H), 5.48-5.67 (m, 2H), 5.08 (d, J = 10.5
Hz, 1H), 4.94 (d, J = 9.5 Hz, 1H), 3.95 (dd, J = 11.3, 3.8 Hz, 2H),
3.53-3.76 (m, 2H), 3.37-3.49 (m, 5H), 3.22-3.37 (m, 2H), 2.35-2.57
(m, 7H), 1.88 (s, 1H), 1.44-1.70 (m, 11H), 1.14-1.39 (m, 8H),
0.72-0.89 (m, 3H) 626.6 46
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(oxan-4-yl)piperazine-1-carboxylate ##STR00164## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 8.48 (d, J = 5.1 Hz, 1H), 7.54 (td, J =
7.7, 1.9 Hz, 1H), 7.09 (d, J = 7.6 Hz, 1H), 7.05 (t, J = 6.0 Hz,
1H), 6.25 (ddd, J = 15.2, 10.8, 1.1 Hz, 1H), 6.04 (d, J = 10.5 Hz,
1H), 5.93 (dd, J = 15.1, 7.5 Hz, 1H), 5.44-5.65 (m, 2H), 5.06 (d, J
= 10.5 Hz, 1H), 4.83 (d, J = 9.3 Hz, 1H), 3.91-4.13 (m, 4H),
3.51-3.76 (m, 3H), 3.42 (br. s., 4H), 2.35-2.59 (m, 3H), 2.30 (s,
1H), 2.22 (br. s., 1H), 1.98 (s, 2H), 1.52-1.77 (m, 10H), 1.32-1.51
(m 9H), 1.10-1.32 (m, 9H), 0.72-0.90 (m, 3H) 650.6 47
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
6-cycloheptyl-2,6-diazaspiro[3.3]heptane-2- carboxylate
##STR00165## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 8.56 (d,
J = 4.9 Hz, 1H), 7.63 (t, J = 7.7 Hz, 1H), 7.18 (d, J = 7.7 Hz,
1H), 7.13 (t, J = 6.1 Hz, 1H), 6.34 (dd, J = 15.3, 10.8 Hz, 1H),
6.13 (d, J = 11.0 Hz, 1H), 6.02 (dd, J = 15.1, 7.5 Hz, 1H),
5.57-5.74 (m, 2H), 5.16 (d, J = 10.8 Hz, 1H), 5.03 (d, J = 9.5 Hz,
1H), 3.92 (br. s., 1H), 3.69-3.83 (m, 3H), 3.53 (d, J = 11.0 Hz,
1H), 2.98 (br. s., 2H), 2.79 (br. s., 2H), 2.48-2.72 (m, 5H), 2.28
(br. s., 1H), 2.01 (br. s., 1H), 1.69-1.79 (m, 7H), 1.66 (br. s.,
1H), 1.44-1.63 (m, 10H), 1.23-1.42 (m, 7H), 1.07 (br. s., 3H),
0.85-0.94 (m, 3H) 652.7 ##STR00166## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 8.56 (d, J = 4.9 Hz, 1H), 7.63 (td, J = 7.7,
1.8 Hz, 1H), 7.18 (d, J = 7.6 Hz, 1H), 7.13 (t, J = 6.1 Hz, 1H),
6.33 (ddd, J = 15.1, 10.9, 1.1 Hz, 1H), 6.13 (d, J = 10.8 Hz, 1H),
6.02 (dd, J = 15.1, 7.5 Hz, 1H), 5.57-5.74 (m, 2H), 5.16 (d, J =
10.5 Hz, 1H), 5.03 (d, J = 9.3 Hz, 1H), 3.68-3.79 (m, 2H),
3.46-3.56 (m, 5H), 2.69-2.77 (m, 1H), 2.49- 2.65 (m, 3H), 2.30 (br.
s., 4H), 1.97- 2.09 (m, 3H), 1.88 (quin, J = 9.1 Hz, 2H), 1.66-1.80
(m, 7H), 1.55 (t, J = 11.9 Hz, 1H), 1.22-1.47 (m, 8H), 0.87-0.93
(m, 3H) 596.6 ##STR00167## .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta.: 8.48 (ddd, J = 5.0, 1.8, 1.0 Hz, 1H), 7.54 (t, J = 7.3 Hz,
1H), 7.09 (d, J = 7.6 Hz, 1H), 7.05 (t, J = 6.1 Hz, 1H), 6.14-6.35
(m, 1H), 6.04 (d, J = 9.8 Hz, 1H), 5.93 (dd, J = 15.1, 7.5 Hz, 1H),
5.46-5.68 (m, 2H), 5.08 (d, J = 10.8 Hz, 1H), 4.94 (d, J = 9.5 Hz,
1H), 3.93 (br. s., 1H), 3.51-3.77 (m, 3H), 3.42 (s, 1H), 2.83 (d, J
= 11.3 Hz, 2H), 2.73 (s, 3H), 2.37-2.60 (m, 3H), 2.20 (s, 3H), 2.04
(br. s., 1H), 1.89-2.00 (m, 3H), 1.81 (br. s., 1H), 1.46-1.73 (m,
10H), 1.14-1.39 (m, 8H), 0.75-0.86 (m, 3H) 584.6 50
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
N-methyl-N-(1-methylpiperidin-4-yl)carbamate ##STR00168## .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta.: 8.48 (d, J = 4.9 Hz, 1H), 7.55
(t, J = 7.7 Hz, 1H), 7.10 (d, J = 8.1 Hz, 1H), 7.05 (t, J = 6.2 Hz,
1H), 6.25 (ddd, J = 15.1, 10.8, 1.0 Hz, 1H), 6.05 (d, J = 10.8 Hz,
1H), 5.93 (dd, J = 15.1, 7.5 Hz, 1H), 5.49-5.67 (m, 2H), 5.23 (s,
1H), 5.08 (d, J = 10.5 Hz, 1H), 4.96 (d, J = 9.3 Hz, 1H), 3.54-3.71
(m, 6H), 3.34-3.48 (m, 5H), 2.39-2.58 (m, 3H), 1.97 (s, 1H), 1.85
(s, 1H), 1.59- 1.70 (m, 5H), 1.47 (t, J = 11.9 Hz, 1H), 1.15-1.39
(m, 8H), 0.77-0.86 (m, 3H) 543.5 51
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
morpholine-4-carboxylate ##STR00169## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 8.48 (d, J = 4.8 Hz, 1H), 7.54 (td, J = 7.7,
1.8 Hz, 1H), 7.02-7.12 (m, 2H), 6.18- 6.31 (m, 1H), 6.06 (s, 1H),
6.03 (s, 1H), 5.93 (dd, J = 15.1, 7.5 Hz, 1H), 5.47-5.67 (m, 2H),
5.08 (d, J = 10.8 Hz, 1H), 4.91 (dd, J = 16.3, 9.5 Hz, 1H), 4.31
(br. s., 1H), 4.22 (br. s., 1H), 3.44-3.70 (m, 4H), 3.42 (br. s.,
1H), 3.35 (d, J = 6.0 Hz, 1H), 3.15 (d, J = 9.8 Hz, 1H), 2.91 (d, J
= 9.0 Hz, 1H), 2.69-2.80 (m, 1H), 2.61-2.69 (m, 1H), 2.32-2.57 (m,
7H), 2.00 (s, 1H), 1.93 (br. s., 1H), 1.82 (d, J = 9.8 Hz, 1H),
1.57-1.73 (m, 7H), 1.46 (t, J = 13.3 Hz, 1H), 1.14-1.39 (m, 8H),
0.73-0.87 (m, 3H) 568.5 52
[(2R,3R,4E,6S,7R,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6R)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
(1S,4R)-5-methyl-2,5-diazabicyclo[2.2.1]heptane-2- carboxylate
##STR00170## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 8.48 (d,
J = 5.1 Hz, 1H), 7.69 (d, J = 5.8 Hz, 1H), 7.36-7.61 (m, 1H),
7.00-7.15 (m, 2H), 6.25 (dd, J = 15.1, 10.8 Hz, 1H), 6.04 (d, J =
10.8 Hz, 1H), 5.93 (dd, J = 15.2, 7.4 Hz, 1H), 5.48-5.69 (m, 2H),
5.08 (d, J = 10.5 Hz, 1H), 4.94 (d, J = 9.0 Hz, 1H), 3.53-3.79 (m,
4H), 3.31-3.53 (m, 2H), 2.36- 2.61 (m, 3H), 2.28 (br. s., 1H), 1.90
(br. s., 3H), 1.66 (s, 6H), 1.60 (br. s., 2H), 1.54 (br. s., 2H),
1.30-1.51 (m, 10H), 1.25 (d, J = 10.5 Hz, 1H), 1.14- 1.22 (m, 3H),
0.80 (d, J = 6.8 Hz, 3H) 664.9 53
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
8-cycloheptyl-3,8-diazabicyclo[3.2.1]octane-3- carboxylate
##STR00171## .sup.1H NMR (400 MHz, CHLOROFORM-d) d ppm 0.90 (d, J =
6.78 Hz, 3 H) 1.24-1.49 (m, 9 H) 1.51-1.64 (m, 2 H) 1.64-1.85 (m, 5
H) 2.01 (br. s.,4 H) 2.43-2.58 (m, 5 H) 2.60-2.77 (m, 4 H)
3.48-3.62 (m, 4 H) 3.62-3.82 (m, 3 H) 4.96- 5.10 (m, 1 H) 5.17 (d,
J = 10.79 Hz, 1 H) 5.58-5.76 (m, 2 H) 6.01 (d, J = 7.53 Hz, 1 H)
6.04 (d, J = 7.53 Hz, 1 H) 6.12 (s, 1 H) 6.15 (s, 1 H) 6.24- 6.43
(m, 1 H) 7.10-7.22 (m, 2 H) 7.63 (td, J = 7.72, 1.88 Hz, 1 H) 8.57
(d, J = 4.85 Hz, 1 H) 570.5
54 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-methyl-1,4-diazepane-1-carboxylate ##STR00172## .sup.1H NMR (400
MHz, CHLOROFORM-d) d ppm 0.80- 1.00 (m, 3 H) 1.13-1.39 (m, 11 H)
1.42-1.48 (m, 3 H) 1.52-1.66 (m, 6 H) 1.66-1.88 (m,7 H) 1.99 (s, 1
H) 2.30 (br. s., 1 H) 2.46-2.66 (m, 7 H) 3.40-3.60 (m, 5 H)
3.64-3.85 (m, 2 H) 5.03 (d, J = 9.29 Hz, 1 H) 5.17 (d, J = 10.79
Hz, 1 H) 5.57-5.75 (m, 2 H) 6.00 (d, J = 7.53 Hz, 1 H) 6.04 (d, J =
7.53 Hz, 1 H) 6.12 (s, 1 H) 6.15 (s, 1 H) 6.27-6.41 (m, 1 H)
7.10-7.22 (m, 2 H) 7.63 (td, J = 7.72, 1.88 Hz, 1 H) 8.57 (d, J =
4.84 Hz, 1 H) 624.7 55 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cyclohexylpiperazine-1-carboxylate ##STR00173## .sup.1H NMR (400
MHz, CHLOROFORM-d) d ppm 0.88 (d, J = 6.78 Hz, 4 H) 1.20-1.46 (m, 7
H) 1.49-1.62 (m, 2 H) 1.66-1.77 (m, 5 H) 1.81 (br. s., 1 H) 1.88
(br. s., 1 H) 2.44-2.66 (m, 3 H) 2.77-2.90 (m, 4 H) 3.02 (s, 1 H)
3.39-3.50 (m, 4 H) 3.61-3.79 (m, 2 H) 5.02 (d, J = 9.54 Hz, 1 H)
5.15 (d, J = 10.54 Hz, 1 H) 5.54-5.74 (m, 2 H) 6.00 (dd, J = 15.06,
7.53 Hz, 1 H) 6.11 (d, J = 10.54 Hz, 1 H) 6.32 (ddd, J = 15.06,
10.79, 1.00 Hz, 1 H) 7.12 (t, J = 6.21 Hz, 1 H) 7.16 (d, J = 8.14
Hz, 1 H) 7.61 (t, J = 7.78 Hz, 1 H) 8.55 (d, J = 4.95 Hz, 1 H)
542.5 ##STR00174## .sup.1H NMR (400 MHz, CHLOROFORM-d) d ppm 0.82-
0.97 (m, 3 H) 1.25-1.37 (m, 4 H) 1.39-1.65 (m, 14 H) 1.75 (s, 7 H)
1.90 (br. s., 1 H) 2.06 (m, 1 H) 2.38 (s, 1 H) 2.49-2.77 (m, 4 H)
2.88 (br. s., 3 H) 3.40-3.62 (m, 3 H) 3.62- 3.89 (m, 4 H) 5.03 (t,
J = 9.41 Hz, 1 H) 5.17 (d, J = 10.54 Hz, 1H) 5.57- 5.76 (m, 2 H)
6.02 (dd, J = 15.31, 7.53 Hz, 1 H) 6.13 (d, J = 10.79 Hz, 1 H) 6.34
(ddd, J = 15.12, 10.73, 1.00 Hz, 1 H) 7.14 (t, J = 6.17 Hz, 1H)
7.18 (d, J = 7.36 Hz, 1 H) 7.28 (s, 2 H) 7.63 (t, J = 7.75 Hz, 1 H)
8.57 (d, J = 4.98 Hz, 1 H) 652.5 ##STR00175## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: ppm 0.81- 0.86 (m, 1 H) 0.88-0.93 (m, 3 H)
1.03 (d, J = 6.78 Hz, 3 H) 1.07-1.17 (m, 1 H) 1.21-1.26 (m, 6 H)
1.28- 1.34 (m, 2 H) 1.40 (t, J = 7.34 Hz, 4 H) 1.48-1.68 (m, 3 H)
1.74 (s, 3 H) 2.29-2.65 (m, 12 H) 3.10 (d, J = 7.28 Hz, 3 H)
3.42-3.57 (m, 7 H) 3.68- 3.78 (m, 1 H) 4.98-5.07 (m, 1 H) 5.12-5.19
(m, 1 H) 5.30 (s, 1 H) 5.54-5.75 (m, 3 H) 6.11 (s, 1 H) 6.28 (s, 1
H) 509.50 58 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-
[(2E,4E,6R)-7-hydroxy-6-methylhepta-2,4-dien-2-
yl]-3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6- yl]
4-methylpiperazine-1-carboxylate ##STR00176## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: ppm 0.86 (s, 1 H) 0.93 (d, J = 6.78 Hz, 2 H)
1.23- 1.49 (m, 9 H) 1.54 (d, J = 11.80 Hz, 1 H) 1.61 (br s, 6 H)
1.65-1.77 (m, 4 H) 1.80 (br s, 2 H) 2.00 (br s, 1 H) 2.49-2.66 (m,
4 H) 2.69 (br s, 3 H) 2.80 (br s, 2 H) 3.32 (s, 1 H) 3.52 (d, J =
6.27 Hz, 1 H) 3.62-3.83 (m, 2 H) 4.15 (br s, 2 H) 5.02 (d, J = 9.29
Hz, 1 H) 5.17 (d, J = 10.79 Hz, 1 H) 5.53- 5.77 (m, 2 H) 6.01 (dd,
J = 15.06, 7.53 Hz, 1 H) 6.13 (d, J = 10.79 Hz, 1 H) 6.33 (ddd, J =
15.12, 10.73, 1.25 Hz, 1 H) 7.14 (t, J = 6.11 Hz, 1 H) 7.18 (d, J =
7.56 Hz, 1 H) 7.28 (s, 3 H) 7.63 (td, J = 7.72, 1.88 Hz, 1 H) 8.57
(d, J = 4.94 Hz, 1 H) 638.34 ##STR00177## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: ppm 0.90 (d, J = 6.78 Hz, 3 H) 1.25 (s, 3 H)
1.33- 1.40 (m, 2 H) 1.41-1.49 (m, 5 H) 1.55-1.62 (m, 4 H) 1.66-1.83
(m, 10 H) 1.99 (s, 1 H) 2.13-2.23 (m, 2 H) 2.43-2.70 (m, 9 H)
2.82-2.95 (m, 2 H) 3.53 (d, J = 10.92 Hz, 1 H) 3.67-3.81 (m, 2 H)
4.02-4.10 (m, 1 H) 4.97-5.07 (m, 1 H) 5.10-5.21 (m, 1 H) 5.57-5.67
(m, 1 H) 5.67- 5.76 (m, 1 H) 5.94-6.07 (m, 1 H) 6.08-6.21 (m, 1 H)
6.26-6.40 (m, 1 H) 7.10-7.22 (m, 2 H) 7.57-7.70 (m, 1 H) 8.53-8.61
(m, 1 H) 686.78 60 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-(8,8-difluoro-3-azabicyclo[3.2.1]octan-3-
yl)piperidine-1-carboxylate
Compounds 61-104 (Table 2) were prepared by the method of Scheme
2.
##STR00178##
General Protocol for the Synthesis of Compounds 61-104:
[0561] Step 1: A solution of E7107 (I, 3.7 g, 5.1 mmol, 1.0 equiv.)
under nitrogen in DMF (100 mL, 0.05M) at 0.degree. C. was treated
with imidazole (2.5 g, 36.1 mmol, 7.0 equiv.) and TBSCl (3.9 g,
25.7 mmol, 5.0 equiv.) was added. The reaction was allowed to warm
to room temperature and stirred for 20 hours, or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
diluted with ethyl acetate and the organic layer was washed with
brine, dried over sodium sulfate, filtered, and concentrated in
vacuo. The resulting oil was purified by silica gel column
chromatography (hexanes/ethyl acetate as eluant) to afford the
desired product (J, 4.7 g, 5.0 mmol, 96%).
[0562] Step 2: To a solution of olefin J (4.7 g, 5.0 mmol, 1.0
equiv.) in THF:H.sub.2O (10:1, 133 mL:13 mL, 0.03M) under nitrogen
at 0.degree. C. was added osmium tetroxide (12.4 mL, 1.0 mmol, 0.2
equiv., 2.5% solution) followed by N-methylmorpholine N-oxide (1.16
g, 9.9 mmol, 2.0 equiv.). The reaction was allowed to warm to room
temperature and stirred for 13 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium sulfite, diluted with ethyl acetate, and the organic
layer was washed with water, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (K, 4.8 g, 4.9 mmol,
99%).
[0563] Step 3: To a solution of diol K (4.4 g, 4.5 mmol, 1.0
equiv.) in benzene (100 mL, 0.05M) under nitrogen at room
temperature was added lead tetraacetate (4.0 g, 9.0 mmol, 2.0
equiv.). The reaction was stirred for 30 minutes, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium sulfite and diluted with dichloromethane.
The organic layer was washed with water, dried over sodium sulfate,
filtered, and concentrated in vacuo. The desired product (L, 1.5 g,
2.3 mmol, 52%) was advanced crude.
[0564] Step 4: To a solution of the corresponding sulfone (2.5
equiv.) in THF (0.02M) under nitrogen at -78.degree. C. was added
KHMDS (2.5 equiv.) dropwise and the reaction was stirred for 10
minutes. Then aldehyde L (1.0 equiv.) in THF (0.5 M) was added
dropwise. The reaction was stirred at -78.degree. C. for five hours
and then allowed to warm to room temperature overnight. The
reaction was quenched with water and diluted with ethyl acetate.
The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product
(M).
[0565] Step 5: A solution of silyl ether M (1.0 equiv.) in Me0H
(0.02M) under nitrogen at room temperature was treated with pTsOH
(2.0 equiv.). The reaction was stirred for 2 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was then diluted with ethyl acetate and washed with brine, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by preparative TLC
(dichloromethane/methanol as eluant) to afford the desired product
(61-104). [0566] Exemplified Protocol for the Synthesis of Compound
63
[0567] Steps 1-3 as above.
[0568] Step 4: To a solution of
(S)-2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl
pyrrolidine-1-carboxylate (45.0 mg, 0.12 mmol, 2.5 equiv.) in THF
(2.0 mL, 0.02M) under nitrogen at -78.degree. C. was added KHMDS
(0.23 mL, 0.12 mmol, 2.5 equiv.) dropwise and the reaction was
stirred for 10 minutes. Then aldehyde L (30.0 mg, 0.05 mmol, 1.0
equiv.) in THF (0.2 mL) was added dropwise. The reaction was
stirred at -78.degree. C. for five hours and then allowed to warm
to room temperature overnight. The reaction was quenched with water
and diluted with ethyl acetate. The organic layer was washed with
water, brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (hexane/ethyl acetate as eluent) to afford
the desired product (M, 35 mg, 0.04 mmol, 76%).
[0569] Step 5: A solution of silyl ether M (35.0 mg, 0.04 mmol, 1.0
equiv.) in MeOH (2.0 mL, 0.02M) under nitrogen at room temperature
was treated with pTsOH (15.0 mg, 0.08 mmol, 2.0 equiv.). The
reaction was stirred for 2 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was then
diluted with ethyl acetate and washed with brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by preparative TLC
(dichloromethane/methanol as eluant) to afford the desired product
(compound 63, 22.2 mg, 32 mmol, 80%). .sup.1H NMR (400 MHz,
METHANOL-d4) .delta.:0.90 (d, J=6.65 Hz, 3 H) 1.09 (d, J=6.78 Hz,
3H) 1.24 (s, 3H) 1.32-1.45 (m, 2H) 1.47-1.85 (m, 15H) 1.85-1.94 (m,
4H) 1.95-2.10 (m, 2H) 2.50-2.68 (m, 4H) 2.96-3.08 (m, 4H) 3.09-3.21
(m, 1H) 3.34-3.39 (m, 4H) 3.52-3.88 (m, 5H) 3.92-4.06 (m, 2H) 4.97
(d, J=9.66 Hz, 1H) 5.07 (d, J=10.67 Hz, 1 H) 5.61 (dd, J=15.18,
9.79 Hz, 1H) 5.72 (d, J=9.79 Hz, 2H) 6.12 (dd, J=10.79, 1.00 Hz,
1H) 6.37 (ddd, J=15.12, 10.85, 0.88 Hz, 1H). MS
(ES+)=688.5[M+H].sup.+.
TABLE-US-00005 TABLE 2 Compounds 61-104 LCMS data Structure,
Compound #, and Chemical Name .sup.1H NMR data (ES+) ##STR00179##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 7.19 (s, 2H),
6.05-6.26 (m, 1H), 5.94-6.05 (m, 1H), 5.45-5.69 (m, 3H), 5.08 (d, J
= 10.5 Hz, 1H), 4.95 (d, J = 9.5 Hz, 1H), 3.67 (br. s., 2H),
3.28-3.54 (m, 8H), 2.36-2.62 (m, 7H), 2.09-2.32 (m, 3H), 1.86 (dt,
J = 13.1, 6.6 Hz, 3H), 1.73-1.81 (m, 3H), 1.55-1.71 (m, 9H),
1.24-1.51 (m, 11H), 1.15-1.22 (m, 4H), 0.97 (d, J = 6.8 Hz, 3H),
0.74-0.91 (m, 3H) 686.5 61
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6S)-6-methyl-9-oxo-9-
pyrrolidin-1-ylnona-2,4-dien-2-yl]-12-oxo-1-
oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine- 1-carboxylate
##STR00180## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.69-0.86
(m, 8 H) 0.93- 1.01 (m, 2 H) 1.09-1.24 (m, 15 H) 1.29 (d, J = 4.52
Hz, 2 H) 1.37-1.53 (m, 5 H) 1.65 (s, 2 H) 1.74 (d, J = 10.79 Hz, 1
H) 1.91 (s, 1 H) 2.11 (t, J = 7.78 Hz, 1 H) 2.33-2.59 (m, 4 H)
2.61-2.73 (m, 1 H) 2.78 (s, 1 H) 2.91-3.07 (m, 1 H) 3.11 (t, J =
7.28 Hz, 1 H) 3.32-3.41 (m, 1 H) 3.44 (br. s., 1 H) 3.60-3.76 (m,
1H) 3.86 (br. s., 1 H) 3.92-4.12 (m, 1 H) 4.47 (s, 1 H) 4.95 (d, J
= 10.79 Hz, 1 H) 5.39 (s, 1 H) 5.40-5.42 (m, 1 H) 5.43-5.70 (m, 2H)
6.00 (d, J = 9.79 Hz, 1 H) 6.18-6.35 (m, 1 H) 62
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6S)-6-methyl-7-
[methyl(propyl)carbamoyl]oxyhepta-2,4-dien-2-yl]-
12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00181## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.65 Hz, 3 H) 1.09 (d, J =
6.78 Hz, 3 H) 1.24 (s, 3 H) 1.32- 1.45 (m, 2 H) 1.47-1.85 (m, 15 H)
1.85-1.94 (m, 4 H) 1.95-2.10 (m, 2 H) 2.50-2.68 (m, 4 H) 2.96-3.08
(m, 4 H) 3.09-3.21 (m, 1 H) 3.34-3.39 (m, 4 H) 3.52-3.88 (m, 5 H)
3.92- 4.06 (m, 2 H) 4.97 (d, J = 9.66 Hz, 1 H) 5.07 (d, J = 10.67
Hz, 1 H) 5.61 (dd, J = 15.18 9.79 Hz, 1 H) 5.72 (d, J = 9.79 Hz, 2
H) 6.12 (dd, J = 10.79, 1.00 Hz, 1 H) 6.37 (ddd, J = 15.12, 10.85,
0.88 Hz, 1 H) 688.5 63 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-
carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1- oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine- 1-carboxylate ##STR00182## .sup.1H NMR
(400 MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.65 Hz, 3 H) 1.10
(d, J = 6.90 Hz, 3 H) 1.24 (s, 3 H) 1.34- 1.47 (m, 2 H) 1.49-2.11
(m, 20 H) 2.12-2.14 (m, 1 H) 2.50-2.68 (m, 4 H) 3.17 (br. s., 4 H)
3.39-3.53 (m, 4 H) 3.61-4.06 (m, 7 H) 4.31-4.45 (m, 1 H) 4.98 (d, J
= 9.66 Hz, 1 H) 5.07 (d, J = 10.67 Hz, 1 H) 5.56-5.81 (m, 3 H) 6.12
(d, J = 10.29 Hz, 1 H) 6.37 (dd, J = 15.18, 10.79 Hz, 1 H) 704.5 64
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-
[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00183## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.91 (d, J = 6.90 Hz, 3 H) 1.10 (d, J =
6.78 Hz, 3 H) 1.20-1.26 (m, 3 H) 1.29-1.43 (m, 4 H) 1.45-1.88 (m,
17 H) 1.91-2.01 (m, 5 H) 2.52-2.65 (m, 4 H) 2.77-2.99 (m, 5 H)
3.36- 3.46 (m, 2 H) 3.46-4.06 (m, 10 H) 4.96 (d, J = 9.54 Hz, 1 H)
5.07 (d, J = 10.67 Hz, 1 H) 5.60 (dd, J = 14.93, 9.91 Hz, 1 H)
5.65-5.78 (m, 2 H) 6.12 (d, J = 11.42 Hz, 1 H) 6.37 (dd, J = 14.87,
10.60 Hz, 1 H) 718.5 65 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-
[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-
1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00184## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.91 (d, J = 6.78 Hz, 3 H) 1.09 (d, J =
6.90 Hz, 3 H) 1.24 (s, 3 H) 1.43- 1.95 (m, 28 H) 2.03-2.10 (m, 2 H)
2.56-2.71 (m, 4 H) 3.19 (br. s., 5 H) 3.36-3.40 (m, 4 H) 3.50 (d, J
= 1.76 Hz, 1 H) 3.63-3.92 (m, 4 H) 3.93- 4.03 (m, 2 H) 4.78 (d, J =
3.39 Hz, 1 H) 4.97 (d, J = 4.77 Hz, 1 H) 4.99 (d, J = 5.77 Hz, 1 H)
5.58-5.82 (m, 3 H) 6.13 (d, J = 10.79 Hz, 1 H) 6.37 (ddd, J =
15.15, 10.89, 0.82 Hz, 1 H) 785.6 66
[(2S,3S,4E,6S,7S,10R)-7-hydroxy-3,7-dimethyl-2-
[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-
carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-10-
(pyrrolidine-1-carbonyloxy)-1-oxacyclododec-4-en- 6-yl]
4-cycloheptylpiperazine-1-carboxylate ##STR00185## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz, 3 H) 1.09 (d, J =
6.78 Hz, 3 H) 1.13-1.21 (m, 3 H) 1.24 (s, 3 H) 1.36-1.73 (m, 13 H)
1.75-1.90 (m, 6 H) 1.92-2.11 (m, 4 H) 2.55 (br. s., 4 H) 3.13 (br.
s., 4 H) 3.21-3.29 (m, 1 H) 3.36-3.42 (m, 2 H) 3.59-4.06 (m, 8 H)
4.98 (d, J = 9.66 Hz, 1 H) 5.07 (d, J = 10.54 Hz, 1 H) 5.56-5.80
(m, 3 H) 6.12 (d, J = 11.17 Hz, 1 H) 6.37 (dd, J = 14.74, 10.73 Hz,
1 H) 702.5 67 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(2S)-2-
methylpyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-
yl]-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00186## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.92 (d, J = 6.78 Hz, 3 H) 1.02-1.14
(m, 6 H) 1.24- 1.27 (m, 3 H) 1.29-1.30 (m, 1 H) 1.33- 1.61 (m, 14
H) 1.64-2.09 (m, 21 H) 2.19-2.33 (m, 1 H) 2.45-2.73 (m, 9 H)
2.80-2.99 (m, 1 H) 3.19-3.41 (m, 1 H) 3.54 (br. s., 7 H) 3.80 (s, 1
H) 3.92- 4.04 (m, 2 H) 5.04 (d, J = 9.41 Hz, 1 H) 5.18 (d, J =
10.67 Hz, 1 H) 5.69 (s, 3 H) 6.11 (d, J = 10.67 Hz, 1 H) 6.29 (dd,
J = 14.93, 10.92 Hz, 1 H) 702.4 68
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(3R)-3-
methylpyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-
yl]-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00187## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.92 (d, J = 6.65 Hz, 3 H) 1.00-1.14
(m, 6 H) 1.25 (s, 3 H) 1.28-1.65 (m, 13 H) 1.67-1.86 (m, 6 H)
1.91-2.13 (m, 3 H) 2.16- 2.32 (m, 1 H) 2.37-2.90 (m, 25 H)
2.94-3.07 (m, 5 H) 3.14-3.41 (m, 3 H) 3.41-3.67 (m, 2 H) 3.80 (br.
s., 5 H) 3.89-4.08 (m, 2 H) 5.03 (d, J = 9.16 Hz, 1 H) 5.18 (d, J =
10.67 Hz, 1 H) 5.70 (d, J = 9.29 Hz, 3 H) 6.06-6.15 (m, 1 H)
6.24-6.35 (m, 1 H) 702.3 69
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(3R)-3-
methylpyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-
yl]-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00188## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.92 (d, J = 6.78 Hz, 3 H) 1.09 (d, J =
6.27 Hz, 3 H) 1.25 (s, 3 H) 1.28-1.42 (m, 2 H) 1.44-1.65 (m 9 H)
1.66-1.81 (m, 6 H) 1.84- 2.05 (m, 5 H) 2.10-2.71 (m, 19 H) 2.80 (t,
J = 4.77 Hz, 4 H) 2.90-3.02 (m, 1 H) 3.51 (s, 2 H) 3.68 (t, J =
4.64 Hz, 4 H) 3.74-3.84 (m, 1 H) 3.74-3.84 (m, 1 H) 3.92-4.13 (m, 2
H) 4.21-4.45 (m, 1 H) 5.03 (d, J = 9.41 Hz, 1 H) 5.16 (d, J = 10.67
Hz, 1 H) 5.42-5.56 (m, 1 H) 5.57-5.82 (m, 3 H) 6.10 (d, J = 11.04
Hz, 1 H) 6.29 (dd, J = 15.06, 11.04 Hz, 1 H) 731.5 70
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2R)-2-
carbamoylpyrrolidine-1-carbonyl]oxy-6-
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00189## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz, 3 H) 1.09 (d, J =
6.78 Hz, 3 H) 1.24 (s, 3 H) 1.96 (br. s., 24 H) 2.54 (d, J = 3.64
Hz, 4 H) 3.10 (br. s., 5 H) 3.36-3.42 (m, 2 H) 3.44-3.53 (m, 1 H)
3.57-3.87 (m, 4 H) 3.89-4.09 (m, 3 H) 4.97 (d, J = 9.66 Hz, 1 H)
5.07 (d, J = 10.79 Hz, 1 H) 5.71 (s, 3 H) 6.11 (d, J = 11.04 Hz, 1
H) 6.37 (dd, J = 14.93, 11.42 Hz, 1 H) 732.4 71
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-2-
[(2E,4E,6S)-7-[(2R)-2-(methoxymethyl)pyrrolidine-
1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00190## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz, 3 H) 1.09 (d, J =
6.78 Hz, 3 H) 1.15 (dd, J = 11.80, 6.40 Hz, 3 H) 1.23 (s, 6 H) 1.58
(d, J = 10.42 Hz, 15 H) 1.76 (s, 5 H) 1.83- 2.21 (m, 4 H) 2.47-2.81
(m, 9 H) 3.98 (d, J = 6.78 Hz, 9 H) 4.96 (d, J = 9.66 Hz, 1 H) 5.07
(d, J = 10.67 Hz, 1 H) 5.58 (dd, J = 15.18, 9.91 Hz, 1 H) 5.64-5.78
(m, 2 H) 6.12 (d, J = 10.67 Hz, 1 H) 6.37 (dd, J = 15.06, 10.79 Hz,
1 H) 716.4 72 [(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2S,5S)-
2,5-dimethylpyrrolidine-1-carbonyl]oxy-6-
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00191## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.90 (d, J = 6.78 Hz, 3 H) 1.09 (d, J =
6.78 Hz, 3 H) 1.24 (s, 3 H) 1.35- 1.44 (m, 2 H) 1.76 (s, 16 H)
1.96- 2.29 (m, 4 H) 2.51-2.71 (m, 4 H) 3.02- 3.14 (m, 4 H)
3.13-3.26 (m, 2 H) 3.40-4.13 (m, 11 H) 4.97 (d, J = 9.66 Hz, 1 H)
5.06 (d, J = 10.79 Hz, 1 H) 5.25 (d, J = 52.70 Hz, 1 H) 5.60 (dd, J
= 15.18, 9.66 Hz, 1 H) 5.65-5.81 (m, 2 H) 6.12 (d, J = 10.42 Hz, 1
H) 6.37 (dd, J = 14.87, 10.85 Hz, 1 H) 706.4 73
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-
fluoropyrrolidine-1-carbonyl]oxy-6-methylhepta-
2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-
1-oxacyclododec-4-en-6-yl] 4- cycloheptylpiperazine-1-carboxylate
##STR00192## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.90 (d,
J = 6.78 Hz, 3 H) 1.09 (d, J = 6.65 Hz, 3 H) 1.24 (s, 3 H) 1.35-
1.46 (m, 2 H) 1.76 (s, 15 H) 1.78- 1.88 (m, 2 H) 1.95-2.07 (m, 3 H)
2.51- 2.69 (m, 4 H) 3.09 (br. s., 4 H) 3.16- 3.25 (m, 2 H)
3.39-4.10 (m, 11 H) 4.98 (d, J = 9.66 Hz, 1 H) 5.07 (d, J = 10.67
Hz, 1 H) 5.25 (dt, J = 53.33, 3.64 Hz, 1 H) 5.61 (dd, J = 15.18,
9.79 Hz, 1 H) 5.72 (m, J = 9.54 Hz, 2 H) 6.12 (d, J = 10.04 Hz, 1
H) 6.31-6.43 (m, 1 H) 706.4 74
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3R)-3-
fluoropyrrolidine-1-carbonyl]oxy-6-methylhepta-
2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-
1-oxacyclododec-4-en-6-yl] 4- cycloheptylpiperazine-1-carboxylate
##STR00193## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.89-0.92
(m, 3 H) 1.06-1.14 (m, 3 H) 1.24 (s, 3 H) 1.34-1.46 (m, 6 H) 1.76
(s, 24 H) 2.55 (br. s., 4 H) 3.06 (br. s., 5 H) 3.13-3.22 (m, 2 H)
3.38- 3.52 (m, 1 H) 3.54-4.12 (m, 8 H) 4.97 (d, J = 9.66 Hz, 1 H)
5.07 (d, J = 10.79 Hz, 1 H) 5.57-5.81 (m, 3 H) 6.12 (d, J = 10.42
Hz, 1 H) 6.31-6.46 (m, 1 H) 716.5 75
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,2-
dimethylpyrrolidine-1-carbonyl)oxy-6-methylhepta-
2,4-dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-
1-oxacyclododec-4-en-6-yl] 4- cycloheptylpiperazine-1-carboxylate
##STR00194## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.72-0.93
(m, 3 H) 1.01 (d, J = 2.26 Hz, 4 H) 1.12- 1.32 (m, 7 H) 1.39-1.56
(m, 8 H) 1.60- 1.72 (m, 5 H) 1.73-1.83 (m, 3 H) 1.99 (d, J = 6.27
Hz, 2 H) 2.38-2.58 (m, 3 H) 2.82 (s, 1 H) 2.90 (d, J = 5.77 Hz, 3
H) 3.02-3.19 (m, 1 H) 3.30 (dt, J = 12.86, 6.24 Hz, 4 H) 3.64-3.77
(m, 4 H) 3.77-3.84 (m, 2 H) 4.94 (d, J = 9.29 Hz, 2 H) 5.01-5.18
(m, 2 H) 5.41 (d, J = 12.30 Hz, 1 H) 5.47-5.67 (m, 2 H) 5.71 (d, J
= 15.31 Hz, 1 H) 6.02 (d, J = 11.29 Hz, 1 H) 6.07-6.25 (m, 1 H)
6.40-6.50 (m, 1 H) 8.24 (br. s., 1 H) 702.4 76
[(2S,3S,4E,6R,7R,10R)-2-[(2E,4E)-6,6-dimethyl-7-
(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-
7,10-dihydroxy-3,7-dimethyl-12-oxo-1- oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine- 1-carboxylate ##STR00195## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.65-0.92 (m, 4 H) 0.94-1.22 (m, 9
H) 1.23-1.33 (m, 3 H) 1.36-1.57 (m, 8 H) 1.59 (br. s., 1 H)
1.61-1.69 (m, 4 H) 1.79 (br. s., 5 H) 1.87-2.04 (m, 3 H) 2.10
(s, 3 H) 2.25 (br. s., 6 H) 2.33-2.52 (m, 5 H) 2.59 (d, J = 10.54
Hz, 2 H) 2.70- 2.86 (m, 3 H) 2.89 (s, 1 H) 2.95 (d, J = 3.51 Hz, 1
H) 2.99-3.07 (m, 1 H) 3.10 (s, 1 H) 3.15 (s, 1 H) 3.22-3.34 (m, 4
H) 3.35-3.52 (m, 1 H) 3.54- 3.68 (m, 3 H) 3.70 (br. s., 1 H) 3.74-
3.98 (m, 2 H) 4.76 (br. s., 1 H) 4.85- 5.02 (m, 2 H) 5.40 (s, 1 H)
5.46-5.75 (m, 3 H) 5.93-6.06 (m, 1 H) 6.07- 6.22 (m, 1 H) 799.48
##STR00196## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.91 (dd,
J = 6.65, 2.76 Hz, 3 H) 1.09 (dd, J = 6.78, 2.26 Hz, 3 H) 1.24 (s,
3 H) 1.36-1.44 (m, 2 H) 1.46-2.10 (m, 22 H) 2.15-2.36 (m, 1 H)
2.51- 2.69 (m, 2 H) 3.15 (d, J = 1.51 Hz, 4 H) 3.23-3.30 (m, 1 H)
3.41-3.60 (m, 2 H) 3.63-4.08 (m, 7 H) 4.21-4.30 (m, 1 H) 4.98 (d, J
= 9.54 Hz, 1 H) 5.07 (d, J = 10.54 Hz, 1 H) 5.61 (dd, J = 15.06,
9.66 Hz, 1 H) 5.71 (s, 2 H) 6.13 (s, 1 H) 6.30-6.45 (m, 1 H) 732.5
78 (2R)-1-[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-
cycloheptylpiperazine-1-carbonyl)oxy-7,10-
dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
en-2-yl]-2-methylhepta-3,5-
dienoxy]carbonylpyrrolidine-2-carboxylic acid ##STR00197## .sup.1H
NMR (400 MHz, METHANOL- d4) .delta.: 0.89 (d, J = 6.78 Hz, 3 H)
1.05- 1.17 (m, 3 H) 1.24 (s, 3 H) 1.33-1.45 (m, 2 H) 1.48-1.89 (m,
16 H) 1.96- 2.10 (m, 2 H) 2.51-2.71 (m, 6 H) 3.12 (br. s., 4 H)
3.59-3.88 (m, 9 H) 4.04 (br. s., 2 H) 4.98 (d, J = 9.66 Hz, 1 H)
5.06 (d, J = 10.67 Hz, 1 H) 5.61 (dd, J = 15.18, 9.79 Hz, 1 H)
5.66-5.79 (m, 2 H) 6.12 (d, J = 10.67 Hz, 1 H) 6.39 (dd, J = 15.18,
10.92 Hz, 1 H) 702.6 79 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6R)-6-methyl-7-(3-
oxopyrrolidine-1-carbonyl)oxyhepta-2,4-dien-2-yl]-
12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00198## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.90 (d, J = 5.90 Hz, 3 H) 1.09 (t, J =
5.65 Hz, 3 H) 1.24 (s, 3H) 1.32- 1.45 (m, 2 H) 1.50-1.89 (m, 16 H)
2.01-2.12 (m, 2 H) 2.20 (q, J = 7.19 Hz, 2 H) 2.53-2.70 (m, 4 H)
3.19 (br. s., 4 H) 3.35-3.44 (m, 2 H) 3.81 (d, J = 5.40 Hz, 7 H)
3.99 (d, J = 6.53 Hz, 2 H) 4.55-4.70 (m, 4 H) 4.98 (d, J = 9.54 Hz,
1 H) 5.07 (d, J = 10.67 Hz, 1 H) 5.72 (d, J = 9.66 Hz, 3 H)
6.08-6.18 (m, 1 H) 6.30-6.43 (m, 1 H) 730.5 80
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-
cycloheptylpiperazine-1-carbonyl)oxy-7,10-
dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
en-2-yl]-2-methylhepta-3,5-dienyl] 2-oxa-7-
azaspiro[3.4]octane-7-carboxylate ##STR00199## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.77-0.89 (m, 6 H) 1.00 (d, J = 6.78
Hz, 3 H) 1.12 (d, J = 6.02 Hz, 1 H) 1.15-1.22 (m,4 H) 1.25-1.28 (m,
1 H) 1.29-1.54 (m, 11 H) 1.55-1.81 (m, 14 H) 1.90 (br. s., 1 H)
1.97 (s, 1 H) 2.37-2.58 (m, 8 H) 3.21-3.38 (m, 5 H) 3.38-3.49 (m, 4
H) 3.56-3.75 (m, 1 H) 3.80-3.98 (m, 2 H) 4.05 (d, J = 7.28 Hz, 1 H)
4.95 (d, J = 9.54 Hz, 1 H) 5.08 (d, J = 10.79 Hz, 1 H) 5.23 (s, 1
H) 5.50-5.69 (m, 3 H) 6.01 (d, J = 10.79 Hz, 1 H) 6.10-6.30 (m, 1
H) 688.65 81 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-
carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1- oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine- 1-carboxylate ##STR00200## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.47-0.61 (m, 2 H) 0.63-0.90 (m, 6
H) 1.02 (s, 1 H) 1.15-1.28 (m, 5 H) 1.30-1.51 (m, 9 H) 1.56 (br.
s., 1 H) 1.59-1.68 (m, 5 H) 1.70-1.87 (m, 5 H) 2.35-2.60 (m, 8 H)
2.63-2.75 (m, 2 H) 2.81 (d, J = 0.75 Hz, 1 H) 2.89 (s, 1 H) 3.17-
3.39 (m, 4 H) 3.42-3.56 (m, 4 H) 3.57- 3.73 (m, 1 H) 3.80 (d, J =
11.04 Hz, 1 H) 3.90-4.13 (m, 2 H) 4.94 (d, J = 9.29 Hz, 1 H) 4.95
(d, J = 9.29 Hz, 1 H) 5.07 (d, J = 10.54 Hz, 1 H) 5.14 (d, J =
10.79 Hz, 1 H) 5.23 (s, 1 H) 5.44-5.66 (m, 3 H) 6.00 (d, J = 11.04
Hz, 1 H) 6.17 (d, J = 10.79 Hz, 1 H) 6.23 (m, J = 10.79 Hz, 1 H)
6.56 (d, J = 11.54 Hz, 1 H) 700.52 ##STR00201## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.91 (d, J = 6.78 Hz, 3 H) 1.10 (d, J =
6.78 Hz, 3 H) 1.24 (s, 3 H) 1.34- 1.45 (m, 2 H) 1.49-1.90 (m, 16 H)
2.00-2.11 (m, 2 H) 2.51-2.70 (m, 4 H) 3.09-3.21 (m, 4 H) 3.23-3.30
(m, 1 H) 3.37 (s, 1 H) 3.54-3.62 (m, 2 H) 3.63-4.04 (m, 7 H) 4.07
(d, J = 3.26 Hz, 2 H) 4.98 (d, J = 9.66 Hz, 1 H) 5.07 (d, J = 10.79
Hz 1 H) 5.57-5.66 (m, 1 H) 5.67-5.79 (m, 2 H) 6.12 (d, J = 10.79
Hz, 1 H) 6.39 (dd, J = 14.74, 10.35 Hz, 1 H) 720.5 83
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3S,4R)-
3,4-dihydroxypyrrolidine-1-carbonyl]oxy-6-
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00202## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.78 (d, J = 6.78 Hz, 3 H) 0.97 (d, J =
6.65 Hz, 3 H) 1.06-1.17 (m, 3 H) 1.23-1.32 (m, 2 H) 1.33-1.76 (m,
16 H) 1.82-1.93 (m, 2 H) 1.94-2.10 (m, 4 H) 2.35-2.57 (m, 4 H)
2.80- 3.07 (m, 6 H) 3.24-3.29 (m, 1 H) 3.31- 3.74 (m, 8 H) 3.86 (d,
J = 6.53 Hz, 2 H) 4.85 (d, J = 9.66 Hz, 1 H) 4.95 (d, J = 10.79 Hz,
1 H) 5.49 (dd, J = 15.18, 9.79 Hz, 1 H) 5.53-5.70 (m, 2 H) 6.00 (d,
J = 10.67 Hz, 1 H) 6.25 (dd, J = 14.87, 11.36 Hz, 1 H) 732.6 84
(3S)-1-[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-
cycloheptylpiperazine-1-carbonyl)oxy-7,10-
dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
en-2-yl]-2-methylhepta-3,5-
dienoxy]carbonylpyrrolidine-3-carboxylic acid ##STR00203## .sup.1H
NMR (400 MHz, METHANOL- d4) .delta.: 0.88 (d, J = 6.78 Hz, 3 H)
1.06 (d, J = 6.90 Hz, 3 H) 1.21 (s, 3 H) 1.31- 1.67 (m, 13 H)
1.69-1.98 (m, 8 H) 2.16 (d, J = 8.53 Hz, 1 H) 2.36 (s, 3 H) 2.39
(s, 3 H) 2.47-2.67 (m, 4 H) 2.75- 3.02 (m, 6 H) 3.11-3.23 (m, 1 H)
3.49-3.74 (m, 6 H) 3.78 (d, J = 3.39 Hz, 1 H) 3.97 (d, J = 6.27 Hz,
2 H) 4.56 (br. s., 2 H) 4.93 (d, J = 9.66 Hz, 1 H) 5.04 (d, J =
10.67 Hz, 1 H) 5.51-5.62 (m, 1 H) 5.63-5.77 (m, 2 H) 6.09 (d, J =
11.29 Hz, 1 H) 6.34 (dd, J = 14.81, 10.79 Hz, 1 H) 731.6 85
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(3S)-3-
(dimethylamino)pyrrolidine-1-carbonyl]oxy-6-
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00204## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.88 (d, J = 6.78 Hz, 3 H) 1.06 (d, J =
6.90 Hz, 3 H) 1.21 (s, 3 H) 1.31- 1.67 (m, 13 H) 1.69-1.98 (m, 8 H)
2.16 (d, J = 8.53 Hz, 1 H) 2.36 (s, 3 H) 2.39 (s, 3 H) 2.47-2.67
(m, 4 H) 2.75- 3.02 (m, 6 H) 3.11-3.23 (m, 1 H) 3.49-3.74 (m, 6 H)
3.78 (d, J = 3.39 Hz, 1 H) 3.97 (d, J = 6.27 Hz, 2 H) 4.56 (br. s.,
2 H) 4.93 (d, J = 9.66 Hz, 1 H) 5.04 (d, J = 10.67 Hz, 1 H)
5.51-5.62 (m, 1 H) 5.63-5.77 (m, 2 H) 6.09 (d, J = 11.29 Hz, 1 H)
6.34 (dd, J = 14.81, 10.79 Hz, 1 H) 686.6 86
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-(2,5-
dihydropyrrole-1-carbonyloxy)-6-methylhepta-2,4-
dien-2-yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1-
oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine- 1-carboxylate
##STR00205## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.90 (d,
J = 6.78 Hz, 3 H) 1.09 (d, J = 6.78 Hz, 3 H) 1.24 (s, 3 H) 1.35-
1.46 (m, 2 H) 1.47-1.73 (m, 11 H) 1.74-2.09 (m, 11 H) 2.50-2.69 (m,
4 H) 2.96-3.07 (m, 4 H) 3.07-3.18 (m, 1 H) 3.37 (s, 2 H) 3.53-3.88
(m, 5 H) 3.92-4.06 (m, 3 H) 4.29-4.51 (m, 2 H) 4.95-5.01 (m, 1 H)
5.04-5.10 (m, 1 H) 5.55-5.81 (m, 3 H) 6.12 (d, J = 10.42 Hz, 1 H)
6.37 (dd, J = 15.00, 10.98 Hz, 1 H) 720.6 87
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E,6R)-7-[(2R)-2-
(fluoromethyl)pyrrolidine-1-carbonyl]oxy-6-
methylhepta-2,4-dien-2-yl]-7,10-dihydroxy-3,7-
dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00206## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.78 (d, J = 6.65 Hz, 3 H) 0.97 (d, J =
6.78 Hz, 3 H) 1.12 (s, 3 H) 1.24- 1.31 (m, 2 H) 1.34 (s, 9 H)
1.38-1.63 (m, 10H) 1.65 (d, J = 0.88 Hz, 3 H) 1.67- 1.78 (m, 3 H)
1.89-2.06 (m, 3 H) 2.40-2.56 (m, 4 H) 3.00-3.12 (m, 5 H) 3.24-4.01
(m, 11 H) 4.86 (d, J = 9.66 Hz, 1 H) 4.95 (d, J = 10.67 Hz, 1 H)
5.50 (m, J = 9.66 Hz, 1 H) 5.54-5.67 (m, 2 H) 6.00 (d, J = 10.67
Hz, 1 H) 6.25 (dd, J = 15.06, 10.79 Hz, 1 H) 803.7 88
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(3S)-3-[(2-
methylpropan-2-yl)oxycarbonylamino]pyrrolidine-
1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-
oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine- 1-carboxylate
##STR00207## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.03-0.02
(m, 1 H) 0.61-0.69 (m, 1 H) 0.83 (d, J = 6.78 Hz, 3 H) 0.99 (d, J =
6.78 Hz, 3 H) 1.16 (s, 3 H) 1.28- 1.37 (m, 2 H) 1.39-1.63 (m, 13 H)
1.64-1.78 (m, 5 H) 1.83-1.95 (m, 2 H) 2.44-2.58 (m, 4 H) 2.76-2.86
(m, 6 H) 2.93 (s, 3 H) 3.28-3.34 (m, 2 H) 3.43-3.77 (m, 7 H)
3.80-3.95 (m, 2 H) 4.87-4.91 (m, 2 H) 4.97-5.02 (m, 1 H) 5.48-5.72
(m, 3 H) 6.01-6.08 (m, 1 H) 6.22-6.33 (m, 1 H) 700.5 89
[(2R,3E,5E)-6-[(2S,3S,4E,6S,7S,10S)-6-(4-
cycloheptylpiperazine-1-carbonyl)oxy-7,10-
dihydroxy-3,7-dimethyl-12-oxo-1-oxacyclododec-4-
en-2-yl]-2-methylhepta-3,5-dienyl] 3-
azabicyclo[3.1.0]hexane-3-carboxylate ##STR00208## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.70-0.93 (m, 4 H) 1.00 (d, J = 6.78
Hz, 1 H) 1.12- 1.21 (m, 5 H) 1.24-1.50 (m, 14H) 1.55- 1.76 (m, 11
H) 1.90 (br. s., 2 H) 2.14 (s, 1 H) 2.36-2.57 (m, 7 H) 3.22-3.43
(m, 5 H) 3.43-3.59 (m, 1 H) 3.68 (br. s., 1 H) 4.94 (d, J = 9.54
Hz, 1 H) 5.08 (d, J = 10.79 Hz, 1 H) 5.44-5.72 (m, 2 H) 5.82 (dd, J
= 15.43, 6.90 Hz, 1 H) 6.03 (d, J = 10.54 Hz, 1 H) 6.09-6.25 (m, 1
H) 7.16-7.21 (m, 3 H) 7.44 (d, J = 7.28 Hz, 1 H) 8.41 (br. s., 2 H)
638.28 90 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E)-6-pyridin-3-ylhepta-
2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00209## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.90 (d, J = 6.78 Hz, 3 H) 1.22 (s, 3
H) 1.27- 1.56 (m, 10 H) 1.45 (d, J = 6.90 Hz, 3 H) 1.65-1.71 (m, 3
H) 1.72 (s, 3 H) 1.79 (m, 2 H) 1.96 (s, 1 H) 2.43-2.64 (m, 8 H)
3.47 (m, 6 H) 3.58-3.83 (m, 2 H) 5.01 (d, J = 9.54 Hz, 1 H) 5.14
(d, J = 10.67 Hz, 1 H) 5.51-5.75 (m, 2 H) 5.99 (dd, J = 15.06, 7.53
Hz, 1 H) 6.11 (d, J = 10.79 Hz, 1 H) 6.25-6.34 (m, 1 H) 7.11 (ddd,
J = 7.43, 4.86, 1.13 Hz, 1 H) 7.16 (d, J = 7.78 Hz, 1 H) 7.60 (td,
J = 7.69, 1.82 Hz, 1 H) 8.54 (d, J = 5.03 Hz, 1 H) 638.5 91
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate ##STR00210## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.89 (d, J = 6.78 Hz, 3 H) 1.23 (s, 3
H) 1.26- 1.55 (m, 10 H) 1.63-1.72 (m, 3 H) 1.73 (s, 3 H) 1.79 (m, 2
H) 1.96 (br. s., 1 H) 2.51 (m, 9 H) 3.49 (s, 4 H) 3.65 (d, J = 6.90
Hz, 2 H) 3.74 (m, 2 H) 5.08 (d, J = 10.92 Hz, 1 H) 5.15 (d, J =
10.67 Hz, 1 H) 5.51-5.64 (m, 1 H) 5.65- 5.76 (m, 1 H) 5.98 (dt, J =
14.74, 7.18 Hz, 1 H) 6.12 (d, J = 10.67 Hz, 1 H) 6.37 (dd, J =
14.93, 10.79 Hz, 1 H) 7.13 (dd, J = 7.47, 5.08 Hz, 1 H) 7.16 (d, J
= 7.78 Hz, 1 H) 7.56-7.66 (m, 1 H) 8.53 (d, J = 4.14 Hz, 1 H) 624.3
92 [(2S,3S,4E,6R,7R,10R)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E)-6-pyridin-2-ylhexa-
2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00211## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 8.01-8.23 (m, 1H), 6.14-6.29 (m, 2H),
6.03 (d, J = 11.5 Hz, 1H), 5.77-5.98 (m, 1H), 5.44-5.68 (m, 2H),
5.23 (s, 1H), 5.08 (d, J = 10.5 Hz, 1H), 4.94 (d, J = 9.3 Hz, 1H),
3.67 (br. s., 2H), 3.31-3.58 (m, 8H), 3.23 (d, J = 3.8 Hz, 1H),
2.37-2.64 (m, 6H), 2.14 (s, 1H), 1.72-1.98 (m, 6H), 1.53-1.71 (m,
8H), 1.37-1.53 (m, 8H), 1.12-1.37 (m, 10H), 0.90-1.07 (m, 1H),
0.70-0.90 (m, 4H) 708.9 93
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E)-6-(2-pyrrolidin-1-
ylpyrimidin-4-yl)hepta-2,4-dien-2-yl]-1- oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine- 1-carboxylate ##STR00212## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.64-0.89 (m, 4 H) 1.07-1.32 (m, 7
H) 1.32- 1.53 (m, 10 H) 1.59-1.70 (m, 5 H) 1.81 (s, 1 H) 1.89 (br.
s., 2 H) 2.33- 2.56 (m, 6 H) 2.71 (br. s., 4 H) 2.76- 3.03 (m, 2 H)
3.42 (s, 1 H) 3.60 (br. s., 3 H) 3.63-3.90 (m, 2 H) 4.93 (d, J =
9.54 Hz, 1H) 5.08 (d, J = 10.79 Hz, 1 H) 5.23 (s, 1 H) 5.49-5.75
(m, 2 H) 5.90 (dd, J = 15.18, 7.65 Hz, 1 H) 6.04 (d, J = 9.79 Hz, 1
H) 6.14-6.31 (m, 1 H) 8.22 (br. s., 1 H) 8.28-8.38 (m, 1 H)
8.38-8.49 (m, 2 H) 639.69
94 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E)-6-pyrazin-2-ylhepta-
2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00213## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 8.03-8.25 (m, 1H), 6.13-6.29 (m, 2H),
6.03 (d, J = 11.5 Hz, 1H), 5.89 (ddd, J = 15.2, 7.9, 4.8 Hz, 1H),
5.48-5.68 (m, 2H), 5.15 (s, 1H), 5.08 (d, J = 10.5 Hz, 1H), 4.77-
5.02 (m, 1H), 3.68 (br. s., 1H), 3.27- 3.53 (m, 4H), 3.08-3.13 (m,
4H), 2.34- 2.58 (m, 6H), 1.89 (br. s., 1H), 1.81 (s, 1H), 1.63-1.75
(m, 4H), 1.60 (br. s., 2H), 1.40-1.54 (m, 11H), 1.37 (br. s., 2H),
1.13-1.34 (m, 8H), 0.70-0.94 (m, 4H) 682.9 95
[(2S,3S,4E,6S,7S,10S)-2-[(2E,4E)-6-[2-
(dimethylamino)pyrimidin-4-yl]hepta-2,4-dien-2-
yl]-7,10-dihydroxy-3,7-dimethyl-12-oxo-1- oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine- 1-carboxylate ##STR00214## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.70-0.92 (m, 4 H) 1.00 (d, J =
6.53 Hz, 1 H) 1.14- 1.21 (m, 4 H) 1.24-1.27 (m, 1 H) 1.29- 1.55 (m,
14 H) 1.55-1.68 (m, 7 H) 1.72 (br. s., 2 H) 1.89 (br. s., 1 H) 2.14
(s, 1 H) 2.21-2.30 (m, 3 H) 2.34-2.60 (m, 7 H) 3.06 (s, 1 H) 3.16
(s, 1 H) 3.42 (br. s., 5 H) 3.59 (t, J = 7.15 Hz, 1 H) 3.67 (br.
s., 1 H) 4.94 (d, J = 9.54 Hz, 1 H) 5.08 (d, J = 10.54 Hz, 1 H)
5.23 (s, 1 H) 5.48-5.74 (m, 2 H) 5.92 (ddd, J = 15.06, 7.53, 4.77
Hz, 1 H) 6.04 (d, J = 10.54 Hz, 1 H) 6.13-6.30 (m, 1 H) 6.78-6.99
(m, 2 H) 8.32 (dd, J = 5.02, 2.26 Hz, 1 H) ##STR00215## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.73-0.88 (m, 4 H) 1.06 (br. s., 1
H) 1.07 (br. s., 1 H) 1.12 (s, 1 H) 1.15-1.27 (m, 7 H) 1.29 (br.
s., 1 H) 1.31-1.49 (m, 11 H) 1.54- 1.76 (m, 12H) 1.89 (br. s., 2 H)
2.19- 2.31 (m, 3 H) 2.34-2.57 (m, 7 H) 2.69 (s, 1 H) 3.06 (s, 1 H)
3.11- 3.27(m, 1 H) 3.27-3.51 (m, 5 H) 3.66 (br. s., 1 H) 3.80 (t, J
= 6.90 Hz, 1 H) 4.94 (d, J = 9.54 Hz, 1 H) 5.07 (d, J = 10.79 Hz, 1
H) 5.23 (s, 1 H) 5.48- 5.71 (m, 2 H) 5.79-6.05 (m, 2 H) 6.07- 6.25
(m, 1 H) 6.88-7.05 (m, 1 H) 7.29-7.41 (m, 1 H) 8.24-8.48 (m, 1 H)
##STR00216## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.68-0.92
(m, 4 H) 1.00 (d, J = 6.78 Hz, 1 H) 1.06 (br. s., 1 H) 1.08 (br.
s., 1 H) 1.12 (br. s., 1 H) 1.14-1.21 (m, 5 H) 1.24-1.50 (m, 15 H)
1.55-1.69 (m, 7 H) 1.73 (br. s., 2 H) 1.78-1.92 (m, 1 H) 1.98 (s, 1
H) 2.14 (s, 1 H) 2.36-2.58 (m, 7 H) 3.06 (s, 1 H) 3.23 (d, J = 4.02
Hz, 1 H) 3.32- 3.50 (m, 5 H) 3.67 (br. s., 1 H) 3.72- 3.89 (m, 1 H)
4.05 (q, J = 7.03 Hz, 1 H) 4.94 (d, J = 9.54 Hz, 1 H) 5.08 (d, J =
10.54 Hz, 1 H) 5.44-5.68 (m, 2 H) 5.94-6.16 (m, 2 H) 6.31 (dd, J =
14.43, 10.92 Hz, 1 H) 6.93 (s, 1 H) 7.01-7.12 (m, 1 H) 7.45 (s, 1
H) 8.57-8.67 (m, 2 H) 639.53 ##STR00217## .sup.1H NMR (400 MHz,
METHANOL- d4) .delta.: 0.77 (dd, J = 12.42, 6.65 Hz, 2 H) 1.06-1.17
(m, 2 H) 1.17-1.28 (m, 7 H) 1.39-1.61 (m 9 H) 1.63-1.71 (m, 3 H)
1.80-1.97 (m, 2 H) 2.06 (s, 1 H) 2.27 (s, 1 H) 2.36-2.60 (m, 2 H)
2.96 (br. s., 3 H) 3.11 (q, J = 7.28 Hz, 4 H) 3.50-3.77 (m, 4 H)
3.86 (t, J = 7.15 Hz, 1 H) 4.85 (d, J = 9.79 Hz, 3 H) 4.94 (d, J =
10.54 Hz, 2 H) 5.39 (s, 1 H) 5.48 (ddd, J = 15.31, 9.79, 2.01 Hz, 1
H) 5.61 (ddd, J = 15.25, 9.60, 1.25 Hz, 1 H) 5.90 (dd, J = 15.06,
7.78 Hz, 1 H) 6.03 (d, J = 10.79 Hz, 1 H) 6.20-6.46 (m, 1 H)
7.12-7.21 (m, 1 H) 7.48-7.69 (m, 2 H) 8.97 (br. s., 1 H) 639.59
##STR00218## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.67-0.94
(m, 5 H) 0.94-1.10 (m, 9 H) 1.14-1.27 (m, 10 H) 1.30-1.50 (m, 15
H)1.53 (br. s., 1 H) 1.58 (br. s., 1 H) 1.61-1.69 (m, 6 H) 1.73 (s,
1 H) 1.79 (d, J = 1.00 Hz, 2 H) 1.86 (d, J = 3.26 Hz, 3 H) 1.97 (s,
1 H) 2.10-2.15 (m, 4H) 2.20-2.32 (m, 2 H) 2.32-2.44 (m, 2 H) 2.51
(br. s., 3 H) 2.71 (br. s., 5 H) 2.87 (br. s., 2 H) 2.98 (q, J =
7.28 Hz, 4 H) 3.05-3.22 (m 2 H) 3.27(d, J = 9.29 Hz, 1 H) 3.36-
3.53 (m, 3 H) 3.53-3.72 (m, 6 H) 3.81 (br. s., 2 H) 4.05 (q, J =
7.03 Hz, 2 H) 4.83-5.02 (m, 3 H) 5.23 (s, 1 H) 5.28-5.50 (m, 3 H)
5.50-5.76 (m, 3 H) 6.29 (s, 1 H) 6.33 (s, 1 H) 6.39 (d, J = 11.04
Hz, 1 H) 6.45-6.61 (m, 1 H) 6.63-6.86 (m, 2 H) 6.93 (s, 1 H) 7.32
(dd, J = 5.40, 1.38 Hz, 1 H) 7.45 (s, 2 H) 8.56 (d, J = 5.52 Hz, 1
H) 8.64 (d, J = 5.27 Hz, 2 H) 9.05 (d, J = 1.00 Hz, 2 H) 9.11-9.30
(m, 2 H) 639.47 ##STR00219## .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta.: 0.72-0.87 (m, 3 H) 1.14-1.21 (m, 3 H) 1.25 (br. s., 1 H)
1.27 (s, 1 H) 1.29-1.53 (m, 12 H) 1.56-1.63 (m, 2 H) 1.65-1.74 (m,
4 H) 1.82-2.00 (m, 1 H) 2.10 (s, 1 H) 2.35-2.56 (m, 7 H) 3.32-3.48
(m, 5 H) 3.56-3.73 (m, 1 H) 3.80 (t, J = 7.15 Hz, 1 H) 4.94 (d, J =
9.29 Hz, 1 H) 5.08 (d, J = 10.54 Hz, 1 H) 5.47- 5.66 (m, 2 H)
5.94-6.13 (m, 2 H) 6.17- 6.35 (m, 1 H) 6.93-7.13 (m, 1 H) 8.59-8.65
(m, 2 H) 639.53 101 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyrimidin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate ##STR00220## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.76-0.88 (m, 3 H) 0.91 (br. s., 1 H)
1.14-1.21 (m, 4 H) 1.24-1.50 (m, 12 H) 1.54-1.75 (m, 7H) 1.84-2.00
(m, 2 H) 2.10 (s, 1 H) 2.33-2.56 (m, 7 H) 3.15 (s, 1 H) 3.33-3.48
(m, 7 H) 3.55-3.72 (m, 1 H) 3.79 (t, J = 7.15 Hz, 1 H) 4.04 (s, 1H)
4.94 (d, J = 9.29 Hz, 1 H) 5.08 (d, J = 10.79 Hz, 1H) 5.47-5.67 (m,
2 H) 5.92-6.13 (m, 2 H) 6.21-6.38 (m, 1 H) 7.06 (t, J = 4.89 Hz, 1
H) 7.45 (s, 1 H) 8.61 (d, J = 4.77 Hz, 2 H) 639.53 102
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E,6S)-6-pyrimidin-2-
ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate ##STR00221## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.70-0.91 (m, 5 H) 1.00 (d, J = 6.53
Hz, 1 H) 1.15- 1.22 (m, 6 H) 1.25 (dd, J = 7.78, 2.51 Hz, 1 H)
1.29-1.49 (m, 14 H) 1.56- 1.75 (m, 9 H) 1.81 (s, 1 H) 1.89 (br. s.,
1 H) 1.98 (s, 1 H) 2.06-2.28 (m, 1 H) 2.35-2.55 (m, 11 H) 3.23 (d,
J = 3.76 Hz, 1 H) 3.39 (br. s., 5 H) 3.54-3.80 (m, 2 H) 4.05 (q, J
= 7.03 Hz, 1 H) 4.94 (d, J = 9.29 Hz, 1 H) 5.08 (d, J = 10.79 Hz, 1
H) 5.23 (s, 1 H) 5.45-5.68 (m, 3 H) 5.96-6.15 (m, 2 H) 6.29 (dd, J
= 15.18, 10.67 Hz, 1 H) 6.91 (dd, J = 5.14, 2.13 Hz, 1 H) 8.41-8.52
(m, 1 H) ##STR00222## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.68-0.91 (m, 4 H) 1.13-1.22 (m, 4 H) 1.25-1.53 (m, 17 H) 1.53-1.76
(m, 9 H) 1.81- 2.00 (m,4 H) 2.35-2.57 (m, 7 H) 3.05- 3.30 (m, 1 H)
3.30-3.49 (m, 8 H) 3.67 (br. s., 1 H) 4.94 (d, J = 9.54 Hz, 1 H)
5.08 (d, J = 10.79 Hz, 1 H) 5.23 (s, 1 H)5.45-5.68 (m, 2 H)
5.92-6.13 (m, 3 H) 6.15-6.42 (m, 2 H) 7.22-7.22 (m, 1 H) 7.22-7.32
(m, 1 H) 707.65 104 [(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-
dimethyl-12-oxo-2-[(2E,4E)-6-(6-pyrrolidin-1-
ylpyridin-2-yl)hepta-2,4-dien-2-yl]-1- oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine- 1-carboxylate
Compounds 105-115 were prepared by the method of Scheme 3.
##STR00223## ##STR00224##
General Protocol for the Synthesis of Compounds 105-115:
[0570] Step 1: A solution of 6-deoxypladienolide D (N, 100.0 mg,
0.2 mmol, 1.0 equiv.) under nitrogen in DMF (8 mL, 0.2M) at
0.degree. C. was treated with imidazole (89.2 mg, 1.3 mmol, 7.0
equiv.) and TBSCl (140.3 mg, 0.9 mmol, 5.0 equiv.). The reaction
was allowed to warm to room temperature and stirred for 20 hours,
or until the reaction was determined to be complete by LCMS or TLC.
The reaction was extracted with ethyl acetate and the organic layer
was washed with brine, dried over sodium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (hexanes/ethyl acetate as eluant) to afford
the desired product (O, 143.0 mg, 0.19 mmol, 100%).
[0571] Step 2: To a solution of olefin O (30.0 mg, 0.04 mmol, 1.0
equiv.) in degassed THF:H.sub.2O (10:1, 1.0 mL:0.1 mL, 0.01M) under
nitrogen at 0.degree. C. was added osmium tetroxide (0.1 mL, 0.008
mmol, 0.2 equiv., 2.5% solution in tent-butanol) followed by
N-methylmorpholine N-oxide (9.2 mg, 0.08 mmol, 2.0 equiv.). The
reaction was allowed to warm to room temperature and stirred for 30
minutes, or until the reaction was determined to be complete by
LCMS or TLC. The reaction was quenched with sodium sulfite, diluted
with ethyl acetate, and the organic layer was washed with water,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(P, 29.2 mg, 0.04 mmol, 93%).
[0572] Step 3: To a solution of triol P (498.2 mg, 0.6 mmol, 1.0
equiv.) in benzene (25 mL, 0.03M) under nitrogen at room
temperature was added lead tetraacetate (553.4 mg, 1.2 mmol, 2.0
equiv.). The reaction was stirred for 30 minutes, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was concentrated and purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product (Q,
232 mg, 0.5 mmol, 80%).
[0573] Step 4: To a solution of the corresponding sulfone (2.5
equiv.) in THF (0.02M) under nitrogen at -78.degree. C. was added
KHMDS (2.5 equiv.) dropwise and the reaction was stirred for 20
minutes. Then aldehyde Q (1.0 equiv.) in THF (0.5 M) was added
dropwise. The reaction was stirred at -78.degree. C. for 90 minutes
and then allowed to warm to -20.degree. C. over 1 hr. The reaction
was quenched with aqueous ammonium chloride solution, diluted with
ethyl acetate, washed with water, brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The resulting oil was
purified by silica gel column chromatography (hexane/ethyl acetate
as eluent) to afford the desired product (R).
[0574] Step 5: To a solution of acetate R (1.0 equiv.) in methanol
(0.1M) at room temperature was added potassium carbonate (2.5
equiv.). The reaction was run for 24 hours, or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
quenched with water, diluted with ethyl acetate, washed with brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil (S) was advanced crude into the next step.
[0575] Step 6: To a solution of alcohol (S) (1.0 equiv.) in
dichloroethane (0.1M) at room temperature was added
N,N-dimethylaminopyridine (0.3 equiv.) followed by 4-nitrophenyl
chloroformate (4.0 equiv.). The reaction was stirred at room
temperature for 24 hours. Next, the corresponding amine (10.0
equiv.) was added at room temperature. After stirring for one hour,
the reaction was concentrated and the resulting oil was purified by
silica gel column chromatography (hexanes/ethyl acetate as eluant)
to afford the desired product (T).
[0576] Step 7: To a solution of silyl ether T in methanol (0.1M) at
room temperature was added p-methoxytoluenesulfonic acid (2.5
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate, diluted with ethyl acetate,
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel column chromatography (hexane/ethyl acetate as eluent) to
afford the desired product (105-115). (Table 3)
Exemplified Protocol for the Synthesis of Compound 114
[0577] Steps 1-3 as above.
[0578] Step 4: To a solution containing
(S)-2-(1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridine
(44.0 mg, 0.1 mmol, 2.5 equiv.) and THF (2.0 mL, 0.02M) under
nitrogen at -78.degree. C. was added KHMDS (0.27 mL, 0.1 mmol, 2.5
equiv.) dropwise and the reaction was stirred for 20 minutes. Then
aldehyde Q (25 mg, 0.05 mmol, 1.0 equiv.) in THF (0.1 mL) was added
dropwise. The reaction was stirred at -78.degree. C. for 90 minutes
and then allowed to warm to -20.degree. C. over 1 hr. The reaction
was quenched with aqueous ammonium chloride solution, diluted with
ethyl acetate, washed with water, brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The resulting oil was
purified by silica gel column chromatography (hexane/ethyl acetate
as eluent) to afford the desired product (R, 21.0 mg, 0.04 mmol,
69%).
[0579] Step 5: To a solution of acetate R (15.2 mg, 0.03 mmol, 1.0
equiv.) in methanol (2 mL, 0.1M) at room temperature was added
potassium carbonate (9.1 mg, 0.07 mmol, 2.5 equiv.). The reaction
was run for 24 hours, or until the reaction was determined to be
complete by LCMS or TLC. The reaction was quenched with water,
diluted with ethyl acetate, washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The resulting oil (S,
14 mg, 0.03 mmol, 100%) was advanced crude into the next step.
[0580] Step 6: To a solution of alcohol (S, 4.2 mg, 0.008 mmol, 1.0
equiv.) in dichloromethane (1 mL, 0.1M) at room temperature was
added N,N-dimethylaminopyridine (0.3 mg, 0.002 mmol, 0.3 equiv.)
followed by 4-nitrophenyl chloroformate (6.4 mg, 0.03 mmol, 4.0
equiv.). The reaction was stirred at room temperature for 24 hours.
Next, N-methyl piperazine (0.009 mL, 0.08 mmol, 10.0 equiv.) was
added at room temperature. After stirring for one hour, the
reaction was concentrated and the resulting oil was purified by
silica gel column chromatography (hexanes/ethyl acetate as eluant)
to afford the desired product (T, 4.9 mg, 0.007 mmol, 94%).
[0581] Step 7: To a solution of silyl ether T (4.9 mg, 0.007 mmol,
1.0 equiv.) in methanol (0.7 mL, 0.1M) at room temperature was
added p-methoxytoluenesulfonic acid (3.6 mg, 0.02 mmol, 2.5
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate, diluted with ethyl acetate,
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel column chromatography (hexane/ethyl acetate as eluent) to
afford the desired product (compound 114, 3.6 mg, 0.007 mmol, 89%).
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.88 (d, J=6.78 Hz,
3H) 0.99 (d, J=6.90 Hz, 3H) 1.13-1.33 (m, 2H) 1.44 (d, J=6.90 Hz,
3H) 1.47-1.51 (m, 1H) 1.73 (d, J=0.75 Hz, 3H) 1.74-1.81 (m, 1 H)
1.84-1.97 (m, 1H) 2.30 (s, 3H) 2.36 (br. s., 4H) 2.39-2.61 (m, 3H)
3.41 (m, 1H) 3.49 (br. s., 4H) 3.67-3.74 (m, 2H) 4.86 (t, J=10.04
Hz, 1H) 5.13 (d, J=10.67 Hz, 1H) 5.35 (dd, J=14.93, 9.66 Hz, 1H)
5.54 (dd, J=15.06, 9.91 Hz, 1H) 6.00 (dd, J=15.12, 7.47 Hz, 1H)
6.12 (d, J=10.92 Hz, 1H) 6.32 (ddd, J=15.09, 10.82, 1.07 Hz, 1H)
7.11 (ddd, J=7.53, 4.89, 1.13 Hz, 1H) 7.16 (d, J=7.91 Hz, 1H) 7.61
(td, J=7.65, 1.88 Hz, 1H) 8.55 (d, J=4.96 Hz, 1H), MS (ES+): 540.3
[M+H].sup.+. [0582] Compound 116 was prepared by the method of
Scheme 4.
##STR00225##
[0583] Step 1: To a solution of intermediate Q (35 mg, 0.062 mmol,
1 equiv) and SPE-11 (23.20 mg, 0.068 mmol, 1.1 equiv) in
tetrahydrofuran (3 mL) at rt under nitrogen atmosphere,
triphenylarsine (18.98 mg, 0.062 mmol, 1 equiv), silver(I) oxide
(71.8 mg, 0.31 mmol, 5 equiv), and
Tris(dibenzylideneacetone)dipalladium (0) (11.35 mg, 0.012 mmol,
0.2 equiv) were added successively and stirred for 16 hr under dark
at the same temperature. The solid was filtered off through Celite
and the pad washed with EtOAc. Excess solvent was removed under
reduced pressure and the obtained residue was purified with silica
gel chromatography (0-50% EtOAc/hexanes) to give the desired
product (SPE-12, 17.6 mg, 0.027 mmol, 43.6%.
[0584] Step 2: To a solution of SPE-12 (17.6 mg, 0.027 mmol, 1
equiv) in THF (2 mL) was added TBAF (0.216 mL, 0.216 mmol, 8 equiv)
at 0.degree. C., then the reaction mixture was gradually warmed up
to room temperature and stirred for 2 hr. The reaction mixture was
dilrectly applied to silica gel and purified by silica gel
chromatography (0-30% EtOAc/hexanes) to give the desired product
(Compound 116, 5.2 mg, 9.69 .mu.mol, 35.8%). .sup.1H NMR (400 MHz,
METHANOL-d4) .delta.: ppm 0.86-1.02 (m, 15H) 1.37 (d, J=3.51 Hz,
8H) 1.47-1.55 (m, 2H) 1.62-1.71 (m, 3H) 1.79 (s, 3H) 1.85-1.96 (m,
2H) 2.02 (s, 3H) 2.42-2.48 (m, 1H) 2.55-2.63 (m, 2H) 2.65-2.76 (m,
1H) 2.83-2.94 (m, 1H) 3.50-3.62 (m, 1H) 3.80 (s, 2H) 4.89-4.97 (m,
1H) 5.01-5.09 (m, 1H) 5.39-5.56 (m, 2H) 5.82-5.96 (m, 1H) 6.11-6.20
(m, 1H) 6.49-6.62 (m, 1H). MS(ES+): 535.56[M-H].sup.-.
TABLE-US-00006 TABLE 3 Compounds 105-116 LCMS data Structure,
Compound #, and Chemical Name .sup.1H NMR data (ES+) ##STR00226##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.89 (d, J = 6.78 Hz,
3 H) 0.99 (d, J = 6.78 Hz, 3 H) 1.06 (d, J = 6.78 Hz, 3 H)
1.16-1.32 (m, 2 H) 1.33-1.55 (m, 10 H) 1.61-1.70 (m, 2 H) 1.73 (s,
3 H) 1.76- 1.92 (m, 7H) 2.41-2.63 (m, 9H) 3.32 (br. s., 2 H)
3.35-3.53 (m, 7 H) 3.71 (s, 1 H, OH) 3.90-4.01 (m, 2 H) 4.86 (t, J
= 10.04 Hz, 1 H) 5.13 (d, J = 10.54 Hz, 1 H) 5.36 (dd, J = 15.00,
9.60 Hz, 1 H) 5.55 (dd, J = 15.00, 9.85 Hz, 1 H) 5.69 (dd, J =
15.12, 7.47 Hz, 1 H) 6.09 (d, J = 10.79 Hz, 1 H) 6.27 (dd, J =
15.12, 10.85 Hz, 1 H) 672.5
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-
(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-e-
n- 6-yl] 4-cycloheptylpiperazine-1-carboxylate ##STR00227## .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta.: 0.90 (d, J = 6.65 Hz, 3 H)
0.99 (d, J = 6.78 Hz, 3 H) 1.06 (d. J = 6.78 Hz, 3 H) 1.16-1.33 (m,
2 H) 1.33- 1.56 (m, 10 H) 1.62-1.70 (m, 2 H) 1.73 (s, 3 H) 1.78
(br. s., 2 H) 1.85-2.08 (m, 3 H) 2.37- 2.63 (m, 9 H) 3.31-3.62 (m,
10 H) 3.71 (s, 1 H, OH) 3.90-4.04 (m, 2 H) 4.42- 4.50 (m, 1 H) 4.85
(t, J = 10.04 Hz, 1 H) 5.13 (d, J = 10.67 Hz, 1 H) 5.31- 5.39 (m, 1
H) 5.55 (dd, J = 14.93, 9.91 Hz, 1 H) 5.69 (dd, J = 15.06, 7.53 Hz,
1 H) 6.09 (d, J = 11.29 Hz, 1 H) 6.20-6.31 (m, 1 H 688.4
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidin-
e-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclodo-
dec-4- en-6-yl] 4-cycloheptylpiperazine-1-carboxylate ##STR00228##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.90 (d, J = 6.78 Hz,
3 H) 0.99 (d, J = 6.78 Hz, 3 H) 1.06 (d, J = 6.78 Hz, 3 H)
1.15-1.35 (m, 2 H) 1.36-1.55 (m, 1 H) 1.73 (s, 3 H) 1.77-1.96 (m, 5
H) 2.30 (s, 3 H) 2.36 (br. s., 4 H) 2.41-2.66 (m, 5 H) 3.24-3.44
(m, 4 H) 3.48 (br. s., 4 H) 3.63 (t, J = 6.90 Hz, 1 H) 3.70 (d, J =
7.15 Hz, 1 H) 3.96 (qd, J = 10.37, 6.78 Hz, 2 H) 4.86 (t, J = 9.98
Hz, 1 H) 5.14 (d, J = 10.67 Hz, 1 H) 5.36 (dd, J = 15.06, 9.66 Hz,
1 H) 5.55 (dd, J = 14.93, 9.91 Hz, 1 H) 5.69 (dd, J = 15.12, 7.47
Hz, 1 H) 6.09 (d, J = 10.79 Hz, 1 H) 6.26 (dd, J = 15.25, 10.10 Hz,
1 H) 590.3
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-
(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-e-
n- 6-yl] 4-methylpiperazine-1-carboxylate ##STR00229## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.90 (d, J = 6.65 Hz, 3 H) 0.99
(d, J = 6.78 Hz, 3 H) 1.07 (d, J = 6.90 Hz, 3 H) 1.19-1.25 (m, 1 H)
1.37-1.54 (m, 1 H) 1.73 (s, 3 H) 1.76-1.83 (m, 1 H) 1.87- 2.05 (m,
3 H) 2.30 (s, 3 H) 2.35 (br. s., 4 H) 2.44-2.63 (m, 5 H) 3.30-3.60
(m, 9 H) 3.72 (m, 2 H) 3.91-4.03 (m, 2 H) 4.46-4.50 (m, 1 H) 4.86
(t, J = 10.04 Hz, 1 H) 5.13 (d, J = 10.54 Hz, 1 H) 5.32-5.40 (dd, J
= 14.93, 10.04 Hz, 1 H) 5.55 (dd, J = 14.93, 9.91 Hz, 1 H) 5.69
(dd, J = 15.31, 7.65 Hz, 1 H) 6.09 (d, J = 10.79 Hz, 1 H) 6.27 (dd,
J = 14.81, 11.04 Hz, 1 H) 606.3
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidin-
e-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclodo-
dec-4-en- 6-yl] 4-methylpiperazine-1-carboxylate ##STR00230##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.90 (d, J = 6.78 Hz,
3 H) 0.99 (d, J = 6.78 Hz, 3 H) 1.07 (d, J = 6.78 Hz, 3 H)
1.15-1.34 (m, 2 H) 1.36-1.53 (m, 1 H) 1.57 (d, J = 6.40 Hz, 1 H)
1.74 (s, 3 H) 1.77-1.93 (m, 3 H) 1.95-2.07 (m, 1 H) 2.42 (s, 3 H)
2.45-2.68 (m, 7 H) 3.17- 3.43 (m, 2 H) 3.45-3.73 (m, 9 H) 3.84-
4.04 (m, 3 H) 4.86 (t, J = 9.98 Hz, 1 H) 5.14 (d, J = 10.67 Hz, 1
H) 5.36 (dd, J = 15.00, 9.60 Hz, 1 H) 5.56 (dd, J = 15.00, 9.98 Hz,
1 H) 5.68 (dd, J = 15.06, 7.28 Hz, 1 H) 6.09 (d, J = 11.04 Hz, 1 H)
6.27 (dd, J = 15.12, 10.85 Hz, 1 H) 620.7
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6R)-7-[(2R)-2-
(hydroxymethyl)pyrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-
- dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1-carboxylate ##STR00231## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.89 (d, J = 6.78 Hz, 3 H) 0.99 (d, J = 6.90
Hz, 3 H) 1.06 (d, J = 6.78 Hz, 3 H) 1.16-1.31 (m, 2 H) 1.38-1.57
(m, 9 H) 1.60-1.70 (m, 3 H) 1.73 (s, 3 H) 1.74- 1.95 (m, 5 H) 1.98
(t, J = 5.02 Hz, 2 H) 2.42-2.64 (m, 9 H) 3.32-3.48 (m, 6 H) 3.71
(s, 1 H) 3.99 (br. s., 3 H) 4.17-4.68 (m, 2 H) 4.86 (t, J = 10.04
Hz, 1 H) 5.13 (d, J = 10.67 Hz, 1 H) 5.36 (dd, J = 15.06, 9.66 Hz,
1 H) 5.55 (dd, J = 15.00, 9.85 Hz, 1 H) 5.60-5.73 (m, 1 H) 6.09 (d,
J = 10.79 Hz, 1 H) 6.27 (dd, J = 14.81, 10.79 Hz, 1 H) 704.6
[(2S,3S,4E,6R,7R,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-10-hydroxy-3,7-dimethyl-12-oxo-1-
- oxacyclododec-4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate
##STR00232## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.89 (d,
J = 6.78 Hz, 3 H) 0.99 (d, J = 6.78 Hz, 3 H) 1.06 (d, J = 6.78 Hz,
3 H) 1.15-1.29 (m, 2 H) 1.47 (br. s., 1 H) 1.73 (s, 3 H) 1.78-1.96
(m, 4 H) 1.99 (t, J = 4.89 Hz, 2 H) 2.49 (s, 3 H) 2.49-2.65 (m, 4
H) 2.68 (br. s., 4 H) 3.39 (m, 2 H) 3.63 (br. s., 4 H) 3.66-3.75
(m, 1 H) 3.87-4.08 (m, 3 H) 4.21-4.69 (m, 3 H) 4.86 (t, J = 10.16
Hz, 1 H) 5.14 (d, J = 10.67 Hz, 1 H) 5.35 (dd, J = 15.06, 9.66 Hz,
1 H) 5.56 (dd, J = 14.93, 10.04 Hz, 1 H) 5.67 (m, 1 H) 6.09 (d, J =
10.67 Hz, 1 H) 6.27 (dd, J = 14.81, 10.54 Hz, 1 H) 622.5
[(2S,3S,4E,6R,7R,10S)-2-[(2E,4E,6R)-7-[(2R)-2-(fluoromethyl)pyrrolidine-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-10-hydroxy-3,7-dimethyl-12-oxo-1-
- oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate
##STR00233## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.86 (d,
J = 6.78 Hz, 3 H) 0.96 (d, J = 6.78 Hz, 3 H) 1.15-1.37 (m, 3 H)
1.45 (d, J = 6.90 Hz, 3 H) 1.74 (s, 3 H) 1.76-1.84 (m, 1H)
1.87-1.96 (m, 1 H) 2.02 (s, 3 H) 2.52 (m, 3 H) 3.41 (d, J = 11.04
Hz, 1 H) 3.69 (m, 2 H) 4.96 (t, J = 10.10 Hz, 1 H) 5.13 (d, J =
10.67 Hz, 1 H) 5.33 (dd, J = 14.93, 9.66 Hz 1 H) 5.54 (dd, J =
14.93, 9.91 Hz , 1 H) 5.99 (dd, J = 14.49, 7.47 Hz, 1 H) 6.11 (d, J
= 10.29 Hz, 1 H) 6.34 (m, 1 H) 7.18 (m, 2 H) 7.56-7.66 (m, 1 H)
8.56 (d, J = 4.27 Hz, 1 H) 455.2
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-
[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-
4-en-6-yl] acetate ##STR00234## .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta.: 0.88 (d, J = 6.78 Hz, 3 H) 0.99 (d, J = 6.78 Hz, 3 H)
1.13-1.33 (m, 3 H) 1.41- 1.54 (m, 9 H) 1.44 (d, J = 7.03 Hz, 3 H)
1.68 (m, 2 H) 1.73 (s, 3 H) 1.78 (m, 2 H) 1.83-1.94 (m, 1 H)
2.38-2.61 (m, 8 H) 3.29-3.55 (m, 5 H) 3.64-3.74 (m, 2 H) 4.85 (t, J
= 10.04 Hz, 1 H) 5.13 (d, J = 10.67 Hz, 1 H) 5.36 (d, J = 9.66 Hz,
1 H) 5.54 (dd, J = 15.00, 9.98 Hz, 1 H) 6.00 (dd, J = 15.06, 7.53
Hz, 1 H) 6.12 (d, J = 11.04 Hz, 1 H) 6.32 (ddd, J = 15.09, 10.82,
1.07 Hz, 1 H) 7.11 (t, J = 6.17 Hz, 1 H) 7.16 (d, J = 7.78 Hz, 1 H)
7.61 (td, J = 7.69, 1.82 Hz, 1 H) 8.55 (d, J = 4.94 Hz, 1 H) 622.4
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-
[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-
4-en-6-yl] 4-cycloheptylpiperazine-1-carboxylate ##STR00235##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.88 (d, J = 6.78 Hz,
3 H) 0.99 (d, J = 6.90 Hz, 3 H) 1.13-1.33 (m, 2 H) 1.44 (d, J =
6.90 Hz, 3 H) 1.47-1.51 (m, 1 H) 1.73 (d, J = 0.75 Hz, 3 H)
1.74-1.81 (m, 1 H) 1.84-1.97 (m, 1 H) 2.30 (s, 3 H) 2.36 (br. s., 4
H) 2.39-2.61 (m, 3 H) 3.41 (m, 1 H) 3.49 (br. s., 4 H) 3.67-3.74
(m, 2 H) 4.86 (t, J = 10.04 Hz, 1 H) 5.13 (d, J = 10.67 Hz, 1 H)
5.35 (dd, J = 14.93, 9.66 Hz, 1 H) 5.54 (dd, J = 15.06, 9.91 Hz, 1
H) 6.00 (dd, J = 15.12, 7.47 Hz, 1 H) 6.12 (d, J = 10.92 Hz, 1 H)
6.32 (ddd, J = 15.09, 10.82, 1.07 Hz, 1 H) 7.11 (ddd, J = 7.53,
4.89, 1.13 Hz, 1 H) 7.16 (d, J = 7.91 Hz, 1 H) 7.61 (td, J = 7.65,
1.88 Hz, 1 H) 8.55 (d, J = 4.96 Hz, 1 H) 540.3
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-
[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-
en-6-yl] 4-methylpiperazine-1-carboxylate ##STR00236## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.87 (d, J = 6.78 Hz, 3 H) 0.99
(d, J = 6.90 Hz, 3 H) 1.14-1.35 (m, 2 H) 1.44 (d, J = 7.03 Hz, 3 H)
1.47-1.51 (m, 1 H) 1.73 (d, J = 1.00 Hz, 3 H) 1.76-1.80 (m, 1 H)
1.87-1.96 (m, 1 H) 2.42-2.61 (m, 3 H) 2.88 (s, 6 H) 3.39 (d, J =
10.92 Hz, 1 H) 3.65-3.77 (m, 2 H) 4.83 (t, J = 10.04 Hz, 1 H) 5.13
(d, J = 10.67 Hz, 1 H) 5.35 (dd, J = 15.00, 9.60 Hz, 1 H) 5.54 (dd,
J = 14.93, 9.91 Hz, 1 H) 5.99 (dd, J = 15.06, 7.53 Hz, 1 H) 6.11
(d, J = 10.92 Hz, 1 H) 6.33 (dd, J = 14.81, 10.92 Hz, 1 H)
7.09-7.15 (m, 1 H) 7.15-7.21 (m, 1 H) 7.57-7.67 (m, 1 H) 8.55 (d, J
= 4.93 Hz, 1 H) 485.2
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-3,7-dimethyl-12-oxo-2-
[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-
4-en-6-yl] N,N-dimethylcarbamate ##STR00237## .sup.1H NMR (400 MHz,
METHANOL-d4) .delta.: ppm 0.86-1.02 (m, 15 H) 1.37 (d, J = 3.51 Hz,
8 H) 1.47-1.55 (m, 2 H) 1.62-1.71 (m, 3 H) 1.79 (s, 3 H) 1.85- 1.96
(m, 2 H) 2.02 (s, 3 H) 2.42-2.48 (m, 1 H) 2.55-2.63 (m, 2 H)
2.65-2.76 (m, 1 H) 2.83-2.94 (m, 1 H) 3.50-3.62 (m, 1 H) 3.80 (s, 2
H) 4.89-4.97 (m, 1 H) 5.01-5.09 (m, 1 H) 5.39-5.56 (m, 2 H)
5.82-5.96 (m, 1 H) 6.11-6.20 (m, 1 H) 6.49-6.62 (m, 1 H) 535.56
[(2S,3S,4E,6R,7R,10S)-10-hydroxy-2-[(2E,4E,6S)-6-hydroxy-7-[(2R,3R)-3-
[(2R,3R)-3-hydroxypentan-2-yl]oxiran-2-yl]-6-methylhepta-2,4-dien-2-yl]-3,-
7- dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl] acetate
Compounds 117-134 were prepared by the method of Scheme 5.
##STR00238## ##STR00239## ##STR00240##
General Protocol for the Synthesis of Compounds 117-134:
[0585] Step 1: To a solution of NaH (8.3 g, 207 mmol, 1.2 equiv.)
in diethyl ether (400 mL, 0.1M) at 0.degree. C. was added
diethyl-2-methylmalonate (U, 30 g, 172 mmol, 1.0 equiv.) dropwise.
The reaction was gradually warmed to reflux and stirred at reflux
for three hours. The reaction was then cooled to room temperature
and iodoform (67.8 g, 172 mmol, 1.0 equiv.) was added dropwise. The
reaction was once again heated at reflux for 24 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was cooled to 0.degree. C., quenched with 10% aqueous hydrochloric
acid, diluted with ether, and washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was then dissolved in ethanol/water/methanol (400 mL,
3:1:1) and KOH (48.3 g, 861 mmol, 5.0 equiv.) was added at room
temperature. The solution was then heated to and maintained at
75.degree. C. for 24 hours. The reaction was cooled to room
temperature and concentrated in vacuo. The resulting oil was
diluted with ethyl acetate and water, extracted into ethyl acetate,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product (V,
26 g, 123 mmol, 71%).
[0586] Step 2: To a solution of acid V (25.0 g, 118 mmol, 1.0
equiv.) in THF (400 mL, 0.3M) at 0.degree. C. was added lithium
aluminum hydride (4.9 g, 130 mmol, 1.1 equiv.). The reaction was
gradually warmed to room temperature and stirred for four hours or
until the reaction was determined to be complete by LCMS or TLC.
The reaction was cooled to 0.degree. C. and quenched with water.
The resulting suspension was charged with Rochelle's salt solution
(20% by volume) and stirred at room temperature for three hours.
The mixture was filtered while washing with ethyl acetate and the
volume of the filtrate was reduced in vacuo. Ethyl acetate was
added and the organic layer was washed with water, brine, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product (W,
15 g, 76 mmol, 64%).
[0587] Step 3: To a solution of alcohol W (60 mg, 0.3 mmol, 1.0
equiv.) in diethyl ether (2 mL, 0.1M) at room temperature was added
manganese dioxide (395 mg, 4.5 mmol, 15.0 equiv.). The reaction was
stirred for two hours or until the reaction was determined to be
complete by LCMS or TLC. The reaction was filtered through
Celite.RTM. and the filtrate was concentrated in vacuo. The crude
product (X, 59 mg, 0.30 mmol, 99%) was advanced without
purification.
[0588] Step 4: To a solution of
(1R,2S)-2-(N-benzyl-2,4,6-trimethylphenylsulfonamido)-1-phenylpropyl
propionate (1.9 g, 4.4 mmol, 1.0 equiv.), prepared according to
literature precedent (Masamune et al. J. Am. Chem. Soc. 1997, 119,
2586-2587) in dichloromethane (40 mL, 0.1M) at -78.degree. C. was
added triethylamine (1.7 ml, 12.3 mmol, 3.0 equiv.) followed by
dropwise addition of
dicyclohexyl(((trifluoromethyl)sulfonyl)oxy)borane (2.67 g, 8.0
mmol, 2.0 equiv.). The reaction was stirred at -78.degree. C. for
two hours. Next, a solution of (E)-3-iodo-2-methylacrylaldehyde (X,
1.2 g, 6.2 mmol, 1.5 equiv.) in dichloromethane (3 mL) was added
dropwise over thirty minutes. The reaction was stirred at
-78.degree. C. for two hours and then allowed to warm to 0.degree.
C. The reaction was quenched with the addition of aqueous hydrogen
peroxide (16 mL, 20.5 mmol) and the reaction was allowed to
gradually warm to room temperature. The solvent volume was reduced
in vacuo and the solution was diluted with dichloromethane and
water. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product (Y,
1.9 g, 2.8 mmol, 69%).
[0589] Step 5: To a solution of alcohol Y (2.8 g, 4.1 mmol, 1.0
equiv.) in dichloromethane (50 mL, 0.1M) at -78.degree. C. was
added 2,6-lutidine (1.0 mL, 8.3 mmol, 2.0 equiv.) followed by
tert-butyldimethylsilyl trifluoromethanesulfonate (1.1 mL, 4.9 mL,
1.2 equiv.). The reaction was gradually warmed to room temperature
and quenched with aqueous ammonium chloride. Ethyl acetate was
added and the organic layer was washed with water, brine, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product (Z,
2.8 g, 3.5 mmol, 85%).
[0590] Step 6: To a solution of ester Z (2.8 g, 3.5 mmol, 1.0
equiv.) in dichloromethane (40 mL, 0.1M) at 0.degree. C. was added
DIBAL (8.9 mL, 8.9 mmol, 2.5 equiv.). The reaction was stirred for
one hour and then quenched with Rochelle's salt solution (20% by
volume) and stirred at room temperature for three hours. The
mixture was filtered through Celite.RTM. while washing with ethyl
acetate and the volume of the filtrate was reduced in vacuo. Ethyl
acetate was added and the organic layer was washed with water,
brine, dried over magnesium sulfate, filtered, and concentrated in
vacuo. The resulting oil was purified by silica gel column
chromatography (hexane/ethyl acetate as eluent) to afford the
desired product (AA, 1.1 g. 2.8 mmol, 80%).
[0591] Step 7: To a solution of alcohol AA (2.97 g, 8.0 mmol, 1.0
equiv.) in dichloromethane (80 mL, 0.1M) at 0.degree. C. was added
Dess-Martin periodinane (4.4 g, 10.4 mmol, 1.3 equiv.). The
reaction was stirred for two hours or until the reaction was
determined to be complete by LCMS or TLC. The reaction was
concentrated in vacuo and the resulting oil was purified by silica
gel column chromatography (hexane/ethyl acetate as eluent) to
afford the desired product (BB, 2.6 g, 7.1 mmol, 88%).
[0592] Step 8: To a solution of methyltriphenylphosphonium bromide
(11.8 g, 33.0 mmol, 3.0 equiv.) in THF (110 mL, 0.1M) at 0.degree.
C. was added n-butyl lithium (13.2 mL, 33.0 mmol, 3.0 equiv.). The
reaction was stirred for 30 minutes and then cooled to -78.degree.
C. Aldehyde BB (4.1, 11.0 mmol, 1.0 equiv.) in THF (0.5 M) was
added dropwise and the reaction was stirred for one hour. The
reaction was quenched with ammonium chloride and warmed to room
temperature. Ethyl acetate was added and the organic layer was
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel column chromatography (hexane/ethyl acetate as eluent) to
afford the desired product (CC, 3.8 g, 10.4 mmol, 94%).
[0593] Step 9: To a solution of olefin CC (0.1 g, 0.4 mmol, 1.0
equiv.) in THF (4 mL, 0.1M) at 0.degree. C. was added TBAF (0.45
mL, 0.4 mmol, 1.1 equiv.). The reaction was stirred for 30 minutes
or until the reaction was determined to be complete by LCMS or TLC.
Diethyl ether was added and the organic layer was washed with
water, brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The crude product (DD, 0.1 g, 0.4 mmol, 99%)
was advanced without purification.
[0594] Step 10: To a solution of alcohol DD (0.15 g, 0.4 mmol, 1.0
equiv.) in dichloromethane (4 mL, 0.1M) at 0.degree. C. was added
EDC (0.10 g, 0.5 mmol, 1.3 equiv.) followed by nonenoic acid (0.08
g, 0.4 mmol, 1.1 equiv.) and DMAP (catalytic). The reaction was
gradually warmed to room temperature and stirred overnight. Ethyl
acetate was added and the organic layer was washed with water,
brine, dried over magnesium sulfate, filtered, and concentrated in
vacuo. The resulting oil was purified by silica gel column
chromatography (hexane/ethyl acetate as eluent) to afford the
desired product (EE, 0.13 g, 0.33 mmol, 81%).
[0595] Step 11: To a solution of ester EE (0.5 g, 1.3 mmol, 1.0
equiv.) in degassed toluene (65 mL, 0.05M) at room temperature was
added benzoquinone (0.007 g, 0.06 mmol, 0.05 equiv.) followed by
Hoyveda-Grubbs catalyst (0.08 g, 0.13, 0.1 equiv.). The reaction
was gradually warmed to 60.degree. C. and stirred overnight. Once
determined to be complete by TLC or LCMS, the reaction was
concentrated. The crude material (FF) was used in the following
step without further purification
[0596] Step 12: To a solution of macrocycle FF (1.0 equiv.) in
dioxane (65 mL, 0.05M) was added selenium dioxide (0.4 g, 3.8 mmol,
3.0 equiv.) at room temperature. The reaction was heated to
80.degree. C. for 3 hours. Ethyl acetate was added, and the organic
layer was washed with water and saturated sodium bicarbonate, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product (GG,
0.3 g, 0.8 mmol, 64%).
[0597] Step 13: To a solution of alcohol GG (1.0 equiv.) in MTBE
(0.1M) at room temperature was added triethylamine (5.0 equiv.),
para-nitrophenylchloroformate (3.0 equiv.), DMAP (catalytic) and
the reaction was stirred overnight. Once determined to be complete
by TLC or LCMS, the reaction was quenched with water. Ethyl acetate
was added and the organic layer was washed with water, saturated
sodium bicarbonate, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The crude product (HH) was advanced without
purification.
[0598] Step 14: To a solution of carbonate HH (1.0 equiv.) in MTBE
(0.1M) at room temperature was added the corresponding amine (2.0
equiv.). Once determined to be complete by TLC or LCMS, the
reaction was concentrated and the resulting oil was purified by
silica gel column chromatography (hexane/ethyl acetate as eluent)
to afford the desired product (II).
[0599] Step 15: To a solution of vinyl iodide II (1.0 equiv.) in
THF (0.1M) at room temperature was added vinyl pinacol boronate
(2.5 equiv.), silver oxide (5.0 equiv.), triphenylarsine (1.2
equiv.), and Pd.sub.2(dba).sub.3 (0.15 equiv.). The reaction was
stirred at room temperature overnight. Once determined to be
complete by TLC or LCMS, the reaction was filtered through
Celite.RTM.. Dichloromethane was added and the organic layer was
washed with water and saturated sodium bicarbonate, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(JJ).
[0600] Step 16: To a solution of diene JJ (1.0 equiv.) in
THF:H.sub.2O (0.1M) at 0.degree. C. was added N-methylmorpholine
N-oxide (1.2 equiv.) and osmium tetroxide in t-BuOH (0.1 equiv.).
The reaction was stirred at room temperature overnight. Once
determined to be complete by TLC or LCMS, the reaction was quenched
by addition of aqueous sodium bicarbonate. Ethyl acetate was added
and the organic layer was washed with water, saturated sodium
bicarbonate, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The crude material (KK) was advanced without
further purification.
[0601] Step 17: To a solution of diol KK (1.0 equiv.) in benzene
(0.1M) at room temperature was added lead tetraacetate (1.2
equiv.). The reaction was stirred at room temperature for 40
minutes or until determined to be complete by TLC or LCMS. The
reaction was quenched by addition of Na.sub.2S.sub.2O.sub.3 and
then sodium bicarbonate. Dichloromethane was added, and the organic
layer was washed with water, saturated sodium bicarbonate, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(LL).
[0602] Step 18: To a solution of the corresponding sulfone (2.5
equiv.) in THF (0.1M) at -78.degree. C. was added KHMDS (2.5
equiv.) and the reaction was stirred at -78.degree. C. for one
hour. Next, a solution of aldehyde LL in THF (1.0 equiv.) was added
dropwise at -78.degree. C. The reaction was allowed to warm
gradually to -20.degree. C. and stirred at -20.degree. C. for two
hours. The reaction was quenched with aqueous sodium bicarbonate
and ethyl acetate was added. The organic layer was washed with
water, saturated sodium bicarbonate, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (117-134).
Exemplified Protocol for the Synthesis of Compound 128
[0603] Steps 1-12 as above.
[0604] Step 13: To a solution of alcohol GG (0.30 g, 0.8 mmol, 1.0
equiv.) in MTBE (3.0 mL, 0.1M) at room temperature was added
triethylamine (0.55 mL, 4.0 mmol, 5.0 equiv.), para-nitrophenyl
chloroformate (0.24 g, 1.2 mmol, 2.0 equiv.), and DMAP (0.12 g, 0.9
mmol, 1.2 equiv.) and the reaction was stirred overnight. Once
determined to be complete by TLC or LCMS, the reaction was quenched
with water. Ethyl acetate was added and the organic layer was
washed with water, saturated sodium bicarbonate, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The crude
product (HH) was advanced without purification.
[0605] Step 14: To a solution of carbonate HH (1.0 equiv.) in MTBE
(0.1M) at room temperature was added N-methylpiperazine (0.13 mL,
1.2 mmol, 1.5 equiv.). Once determined to be complete by TLC or
LCMS, the reaction was concentrated and the resulting oil was
purified by silica gel column chromatography (hexane/ethyl acetate
as eluent) to afford the desired product (II, 0.30 g, 0.5 mmol,
67.5%).
[0606] Step 15: To a solution of vinyl iodide II (0.15 g, 0.30
mmol, 1.0 equiv.) in THF (3.0 mL, 0.1M) at room temperature was
added vinyl pinacol boronate (0.13 mL, 0.30 mmol, 2.5 equiv.),
silver oxide (0.35 g, 1.50 mmol, 5.0 equiv.), triphenylarsine (0.11
g, 0.36 mmol, 1.2 equiv.), and Pd.sub.2(dba).sub.3 (0.04 g, 0.04
mmol, 0.15 equiv.). The reaction was stirred at room temperature
overnight. Once determined to be complete by TLC or LCMS, the
reaction was filtered through Celite.RTM.. Dichloromethane was
added and the organic layer was washed with water, saturated sodium
bicarbonate, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (dichloromethane/methanol as eluent) to
afford the desired product (JJ, 0.12 g, 0.3 mmol, 96%).
[0607] Step 16: To a solution of diene JJ (0.12 g, 0.3 mmol, 1.0
equiv.) in THF:H.sub.2O (4 mL:0.4mL, 0.1M) at 0.degree. C. was
added N-methylmorpholine N-oxide (0.04 g, 0.35 mmol, 1.2 equiv.)
and osmium tetroxide in t-BuOH (0.37 mL, 0.03 mmol, 0.1 equiv.).
The reaction was stirred at room temperature overnight. Once
determined to be complete by TLC or LCMS, the reaction was quenched
by addition of aqueous sodium bicarbonate. Ethyl acetate was added
and the organic layer was washed with water, saturated sodium
bicarbonate, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The crude material (JJ, 0.13 g, 0.3 mmol,
100%) was advanced without further purification.
[0608] Step 17: To a solution of diol KK (0.10 g, 0.23 mmol, 1.0
equiv.) in acetone (3.0 mL, 0.1M) at room temperature was added
diacetoxyiodobenzene (0.12 g, 0.27 mmol, 1.2 equiv.). The reaction
was stirred at room temperature for 40 minutes or until determined
to be complete by TLC or LCMS. The reaction was quenched by
addition of Na.sub.2S.sub.2O.sub.3 and then sodium bicarbonate.
Dichloromethane was added, and the organic layer was washed with
water, saturated sodium bicarbonate, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (LL, 0.05 g, 0.11 mmol,
49%).
[0609] Step 18: To a solution of
(S)-2-(1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridine
(0.08 g, 0.25 mmol, 2.5 equiv.) in THF (0.6 mL, 0.1M) at
-78.degree. C. was added KHMDS (0.50 mL, 0.25 mmol, 2.5 equiv.) and
the reaction was stirred at -78.degree. C. for one hour. Next, a
solution of aldehyde LL (0.04 g, 0.1 mmol, 1.0 equiv.) in THF (0.1
mL.) was added dropwise at -78.degree. C. The reaction was allowed
to warm gradually to -20.degree. C. and stirred at -20.degree. C.
for two hours. The reaction was quenched with aqueous sodium
bicarbonate and ethyl acetate was added. The organic layer was
washed with water, saturated sodium bicarbonate, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(compound 128, 0.03 g, 0.06 mmol, 60%). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.87 (d, J=6.76 Hz, 3H) 1.25 (br. s, 4H)
1.43 (d, J=6.8 Hz, 3H) 1.60 (br. s., 4H) 1.76-1.85 (m, 3H) 2.22 (m,
1H) 2.31 (br. s, 4H), 2.37 (br. s, 4H) 2.45-2.53 (m, 1H) 3.48 (br.
s., 4H) 3.70 (m, 1H) 4.99-5.12 (m, 2H) 5.36 (m, 1H) 5.43-5.51 (m,
1H) 5.98 (dd, J=15.06, 7.53 Hz, 1H) 6.13 (d, J=11.17 Hz, 1 H) 6.32
(ddd, J=15.06, 10.92, 1.13 Hz, 1H) 7.11 (t, J=6.14 Hz, 1H) 7.16 (d,
J=8.08 Hz, 1H) 7.61 (td, J=7.69, 1.82 Hz, 1H) 8.54 (d, J=4.96 Hz,
1H). MS (ES+)=510.1 [M+H].sup.+.
TABLE-US-00007 TABLE 4 Compounds 117-134 LCMS data Structure,
Compound #, and Chemical Name .sup.1H NMR data (ES+) ##STR00241##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.84 (d, J = 6.78 Hz,
3 H) 1.00 (d, J = 6.78 Hz, 3 H) 1.20 (d, J = 2.76 Hz, 3 H)
1.37-1.60 (m, 12 H) 1.62-1.72 (m, 6 H) 1.72-1.84 (m, 7 H) 1.89-2.33
(m, 19 H) 2.38-2.61 (m, 3 H) 2.74 (br. s., 4 H) 2.90-3.02 (m, 1 H)
3.30 (m, J = 6.02, 6.02 Hz, 4 H) 3.56-3.67 (m, 3 H) 3.90 (qd, J =
10.35, 6.71 Hz, 2 H) 4.91-5.08 (m, 2 H) 5.25-5.36 (m, 1 H)
5.38-5.47 (m, 1 H) 5.62 (dd, J = 15.12, 7.47 Hz, 1 H) 6.04 (dd, J =
10.79, 1.00 Hz, 1 H) 6.21 (ddd, J = 15.31, 10.79, 1.00 Hz, 1 H)
642.4
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-
carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4- cycloheptylpiperazine-1-carboxylate ##STR00242## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.89 (d, J = 6.8 Hz, 3 H) 1.06 (d,
J = J = 6.8 Hz, 3 H) 1.06 (d, J = 6.8 Hz, 3 H) 1.18-1.36 (m, 4 H)
1.41-1.67 (m, 4 H) 1.73 (m, 3 H) 1.84 (m, 6 H) 2.12-2.27 (m, 1 H)
2.32 (br. s, 4 H) 2.38 (br. s, 4H) 2.43-2.63 (m, 2 H) 3.26-3.44 (m,
4 H) 3.49 (br. s., 4 H) 3.91-4.01 (m, 2 H) 4.12 (d, J = 7.15 Hz, 1
H) 5.02 (d, J = 10.6 Hz, 1H) 5.06-5.13 (m, 1 H) 5.37 (dd, J =
14.93, 9.41 Hz, 1 H) 5.48 (dd, J = 15.06, 9.66 Hz, 1 H) 5.67 (dd, J
= 15.12, 7.47 Hz, 1 H) 6.10 (d, J = 10.79 Hz, 1 H) 6.23-6.31 (m, 1
H) 560.4
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-(pyrrolidine-1-
carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-en-6-yl]
4- methylpiperazine-1-carboxylate ##STR00243## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.88 (d, J = 6.76 Hz, 3 H) 1.06 (d, J =
6.8 Hz, 3 H) 1.25 (m, 4 H) 1.45-1.63 (m, 4 H) 1.73 (s, 3 H)
1.79-1.89 (m, 6 H) 2.18-2.25 (m, 1 H) 2.29-2.37 (m, 1 H) 2.45-2.63
(m, 2 H) 2.87 (s, 6 H) 3.36 (br. s., 4 H) 3.91-4.00 (m, 2 H) 5.00
-5.11 (m, 2 H) 5.34-5.51 (m, 2 H) 5.67 (dd, J = 15.12, 7.34 Hz, 1
H) 6.10 (d, J = 10.92 Hz, 1 H) 6.23-6.31 (m, 1 H) 505.5, 527.4
[(2R,3E,5E)-6-[(2S,3S,4E,6R)-6-(dimethylcarbamoyloxy)-3-methyl-12-
oxo-1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl]
pyrrolidine- 1-carboxylate ##STR00244## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.89 (d, J = 6.78 Hz, 3 H) 1.08 (d, J = 6.8
Hz, 3 H) 1.14 (s, 1 H) 1.20-1.33 (m, 4 H) 1.46-1.63 (m, 4 H) 1.73
(s, 3 H) 1.77-1.90 (m, 2 H) 1.90-2.04 (m, 2 H) 2.24-2.26 (m, 1 H)
2.29-2.38 (m, 1 H) 2.46-2.64 (m, 2 H) 2.87 (s, 6 H) 3.52 (br. s., 4
H) 3.92-4.02 (m, 2 H) 4.43- 4.50 (m, 1 H) 4.98-5.11 (m, 2 H) 5.35-
5.51 (m, 2 H) 5.67 (dd, J = 15.18, 7.65 Hz, 1 H) 6.10 (d, J = 11.04
Hz, 1 H) 6.23-6.31 (m, 1 H) 543.3
[(2R,3E,5E)-6-[(2S,3S,4E,6R)-6-(dimethylcarbamoyloxy)-3-methyl-12-oxo-
1-oxacyclododec-4-en-2-yl]-2-methylhepta-3,5-dienyl] (3R)-3-
hydroxypyrrolidine-1-carboxylate ##STR00245## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.89 (d, J = 6.76 Hz, 2 H) 1.06 (d, J = 6.8
Hz, 2 H) 1.19-1.33 (m, 4 H) 1.41 (br. s., 1 H) 1.47-1.56 (m, 4 H)
1.61 (br. s., 2 H) 1.73 (s, 3 H) 1.79-1.87 (m, 2 H) 1.87-2.04 (m, 2
H) 2.22-2.24 (m, 1 H) 2.33 (br. s, 4H), 2.39 (br. s, 4 H) 2.47-
2.53 (m, 1 H) 2.56-2.63 (m, 1 H) 3.41- 3.57 (m, 8 H) 3.91-4.02 (m,
2 H) 4.45- 4.49 (m, 1 H) 4.98-5.13 (m, 2 H) 5.37 (dd, J = 15.06,
9.41 Hz, 1 H) 5.48 (dd, J = 15.00, 9.60 Hz, 1 H) 5.67 (dd, J =
15.12, 7.22 Hz, 1 H) 6.10 (d, J = 11.29 Hz, 1 H) 6.23-6.31 (m, 1 H)
576.4
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-carbonyl]oxy-
6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-
yl] 4-methylpiperazine-1-carboxylate ##STR00246## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.88 (d, J = 6.72 Hz, 2 H) 1.06 (d, J =
6.76 Hz, 2 H) 1.14 (br. s., 2 H) 1.19 (br. s, 2 H) 1.26 (br. s., 4
H) 1.47 (br. s, 1 H) 1.54 (s, 3 H) 1.57 (br. s, 4 H) 1.72 (s, 3 H)
1.76-1.91 (m, 4 H) 1.93-2.03 (m, 1 H) 2.2-2.4 (m, 1 H) 2.31 (br. s,
4 H) 2.38 (br. s., 4 H) 2.41-2.63 (m, 2 H) 3.40 (br. s., 2 H) 3.49
(br. s., 4 H) 3.86- 4.04 (m, 3 H) 5.02 (d, J = 10.68 Hz, 1H)
5.06-5.13 (m, 1 H) 5.34-5.42 (m, 1 H) 5.44-5.52 (m, 1 H) 5.66 (dd,
J = 15.00, 7.97 Hz, 1 H) 6.10 (d, J = 11.04 Hz, 1 H) 6.27 (dd, J =
14.81, 11.04 Hz, 1 H) 574.6
[(2S,3S,4E,6R)-3-methyl-2-[(2E,4E,6R)-6-methyl-7-[(2S)-2-
methylpyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-
oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate
##STR00247## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.88 (d,
J = 6.76 Hz, 3 H) 1.06 (d, J = 6.8 Hz, 3 H) 1.25 (m, 4 H) 1.43-1.67
(m, 4 H) 1.73 (s, 3 H) 1.77-1.89 (m, 3 H) 1.96-2.03 (m, 1 H)
2.22-2.25 (m, 1 H) 2.31 (br. s, 4H) 2.37 (br. s, 4 H) 2.47- 2.53
(m, 1H) 2.58-2.64 (m, 1H) 3.32- 3.42 (m, 1 H) 3.44-3.69 (m, 5 H)
2.58- 3.66 (m, 2H) 3.92-4.03 (m, 3 H) 5.02 (d, J = 10.68 Hz, 1H),
5.06-5.13 (m, 1 H) 5.30-5.51 (m, 2 H) 5.66 (dd, J = 15.18, 7.53 Hz,
1 H) 6.10 (d, J = 10.92 Hz, 1 H) 6.27 (dd, J = 15.12, 10.85 Hz, 1
H) 590.5
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-
oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate
##STR00248## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.89 (d,
J = 6.76 Hz, 3 H) 1.06 (d, J = 6.8 Hz, 3 H) 1.26 (br. s, 4 H)
1.38-1.63 (m, 13 H) 1.64-1.77 (m, 6 H) 1.80 (m, 2 H) 1.88-2.05 (m,
2 H) 2.17-2.26 (m, 1 H) 2.28-2.40 (m, 1 H) 2.42-2.63 (m, 6 H)
3.33-3.56 (m, 8 H) 3.84-4.03 (m, 2 H) 4.48 (br. s, 1 H) 4.97-5.12
(m, 3 H) 5.31-5.51 (m, 2 H) 5.67 (dd, J = 15.06, 7.03 Hz, 1 H) 6.09
(d, J = 10.79 Hz, 1 H) 6.17-6.38 (m, 1 H) 658.5
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-carbonyl]oxy-6-
-
methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4- cycloheptylpiperazine-1-carboxylate ##STR00249## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 6.12-6.31 (m, 1H), 6.04 (m, 1H),
5.59 (dd, J = 15.1, 7.5 Hz, 1H), 5.36-5.46 (m, 1H), 5.23-5.36 (m,
1H), 4.90-5.08 (m, 2H), 4.31 (d, J = 5.3 Hz, 1H), 3.84-3.99 (m,
3H), 3.81 (br. s., 1H), 3.69-3.78 (m, 1H), 3.50-3.66 (m, 2H),
3.36-3.47 (m, 4H), 3.17-3.35 (m, 1H), 2.91 (q, J = 9.5 Hz, 2H),
2.40-2.60 (m, 5H), 2.08-2.35 (m, 2H), 1.86-2.02 (m, 1H), 1.64-1.82
(m, 6H), 1.38-1.64 (m, 6H), 0.94-1.08 (m, 3H), 0.82 (d, J = 6.8 Hz,
3H) 658.8
[(2S,3S,4E,6R)-2-[(2E,4E,6R)-7-[(2R)-2-(hydroxymethyl)pyrrolidine-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-
oxacyclododec-4-en-6-yl]
4-(2,2,2-trifluoroethyl)piperazine-1-carboxylate ##STR00250##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.86 (d, J = 6.78 Hz,
3 H) 1.21-1.31 (m, 4 H) 1.44 (d, J = 7.03 Hz, 3 H) 1.47- 1.60 (m, 2
H) 1.73 (d, J = 1.00 Hz, 3 H) 1.76-1.91 (m, 2 H) 2.00 (s, 3 H) 2.22
(dt, J = 13.87, 4.80 Hz, 1 H) 2.33 (ddd, J = 13.77, 11.95, 4.14 Hz,
1 H) 2.43-2.54 (m, 1 H) 3.70 (quin, J = 6.93 Hz, 1 H) 5.02 (d, J =
10.67 Hz, 1 H) 5.17 (td, J = 10.07, 4.96 Hz, 1 H) 5.35 (dd, J =
14.93, 9.54 Hz, 1 H) 5.48 (dd, J = 14.93, 9.66 Hz, 1 H) 5.98 (dd, J
= 15.12, 7.59 Hz, 1 H) 6.12 (d, J = 10.92 Hz, 1 H) 6.32 (ddd, J =
15.09, 10.82, 1.07 Hz, 1 H) 7.11 (ddd, J = 7.53, 4.89, 1.13 Hz, 1
H) 7.16 (d, J = 7.91 Hz, 1 H) 7.61 (td, J = 7.69, 1.82 Hz, 1 H)
8.55 (d, J = 4.88 Hz, 1 H) 426.1
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-pyridin-
2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] acetate
##STR00251## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.87 (d,
J = 6.90 Hz, 4 H) 1.20-1.33 (m, 10 H) 1.44 (d, J = 7.03 Hz, 3 H)
1.46-1.69 (m, 10 H) 1.73 (s, 3 H) 1.79 (m, 4 H) 2.17-2.25 (m, 1 H)
2.27-2.39 (m, 1 H) 2.47 (m, 5 H) 3.07-3.27 (m, 1 H) 3.43 (br. s., 4
H) 3.70 (quin, J = 7.03 Hz, 1 H) 4.99-5.03 (m, 1 H) 5.08 (td, J =
9.82, 4.45 Hz, 1 H) 5.32-5.40 (m, 1 H) 5.42-5.51 (m, 1 H) 5.97 (dd,
J = 15.06, 7.40 Hz, 1 H) 6.12 (d, J = 11.42 Hz, 1 H) 6.28-6.36 (m,
1 H) 7.11 (ddd, J = 7.53, 4.89, 1.13 Hz, 1 H) 7.16 (d, J = 7.91 Hz,
1 H) 7.61 (t, J = 7.65 Hz, 1 H) 8.54 (d, J = 4.88 Hz, 1 H) 592.3
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec- 4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate ##STR00252## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.87 (d, J = 6.76 Hz, 3 H) 1.25 (br. s,
4H) 1.43 (d, J = 6.8 Hz, 3 H) 1.60 (br. s., 4 H) 1.76-1.85 (m, 3 H)
2.22 (m, 1 H) 2.31 (br. s, 4H), 2.37 (br. s, 4H) 2.45- 2.53 (m, 1H)
3.48 (br. s., 4 H) 3.70 (m, 1 H) 4.99-5.12 (m, 2 H) 5.36 (m, 1 H)
5.43-5.51 (m, 1 H) 5.98 (dd, J = 15.06, 7.53 Hz, 1 H) 6.13 (d, J =
11.17 Hz, 1 H) 6.32 (ddd, J = 15.06, 10.92, 1.13 Hz, 1 H) 7.11 (t,
J = 6.14 Hz, 1 H) 7.16 (d, J = 8.08 Hz, 1 H) 7.61 (td, J = 7.69,
1.82 Hz, 1 H) 8.54 (d, J = 4.96 Hz, 1 H) 510.1
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec- 4-en-6-yl]
4-methylpiperazine-1-carboxylate ##STR00253## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.87 (d, J = 6.76 Hz, 3 H) 1.25 (br. s, 4H)
1.43 (d, J = 6.8 Hz, 3H) 1.46-1.64 (m, 2 H) 1.73 (s, 3H) 1.76-1.86
(m, 2 H) 2.21 (m, 1 H) 2.27-2.37 (m, 1 H) 2.43- 2.54 (m, 1 H) 2.87
(s, 6 H) 3.71 (t, J = 7.03 Hz, 1 H) 4.99-5.10 (m, 2 H) 5.34- 5.50
(m, 2 H) 5.97 (dd, J = 15.06, 7.53 Hz, 1 H) 6.12 (d, J = 10.92 Hz,
1 H) 6.32 (dd, J = 15.18, 10.79 Hz, 1 H) 7.11 (t, J = 6.13 Hz, 1 H)
7.17 (d, J = 7.52 Hz, 1 H) 7.61 (t, J = 7.25 Hz, 1 H) 8.54 (d, J =
4.96 Hz, 1 H) 455.2 [(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E,6S)-6-
pyridin-2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec- 4-en-6-yl]
N,N-dimethylcarbamate ##STR00254## 636.9
[(2S,3S,4E,6R)-2-[(2E,4E)-6-[2-(dimethylamino)pyrimidin-4-
yl]hepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-cycloheptylpiperazine-1-carboxylate ##STR00255## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 8.15-8.34 (m, 1H), 7.39 (s, 1H), 6.23-
6.42 (m, 2H), 6.14 (d, J = 11.3 Hz, 1H), 5.97 (ddd, J = 15.1, 7.8,
3.9 Hz, 1H), 5.30-5.58 (m, 2H), 4.94-5.18 (m, 2H), 3.60 (t, J = 6.3
Hz, 3H), 3.29-3.56 (m, 4H), 2.51 (d, J = 6.8 Hz, 4H), 2.30-2.44 (m,
1H), 2.16-2.29 (m, 1H), 1.91-2.11 (m, 4H), 1.70-1.89 (m, 6H), 1.64
(br. s., 5H), 1.45-1.62 (m, 8H), 1.34-1.44 (m, 4H), 1.18-1.33 (m,
4H), 1.10 (br. s., 1H), 0.78-1.02 (m, 3H) 662.9
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-(2-pyrrolidin-1-
ylpyrimidin-4-yl)hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] 4-
cycloheptylpiperazine-1-carboxylate ##STR00256## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 8.03 (dd, J = 5.3, 1.8 Hz, 1H), 7.20
(s, 1H), 6.05-6.23 (m, 2H), 5.96 (d, J = 10.8 Hz, 1H), 5.74-5.87
(m, 1H), 5.13-5.38 (m, 2H), 4.82-5.01 (m, 2H), 4.35 (d, J = 4.3 Hz,
1H), 3.43-3.60 (m, 3H), 3.34 (d, J = 3.3 Hz, 1H), 3.20-3.31 (m,
4H), 2.42 (br. s., 1H), 2.33 (br. s., 4H), 2.01-2.27 (m, 3H),
1.72-1.96 (m, 3H), 1.65 (br. s., 6H), 1.58 (s, 4H), 1.51 (d, J =
7.0 Hz, 2H), 1.18-1.44 (m, 14H), 0.96-1.14 (m, 4H), 0.71-0.85 (m,
11H), 0.46 (q, J = 7.9 Hz, 5H) 793.3
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-[2-[(3S)-3-triethylsilyloxypyr-
rolidin-1-
yl]pyrimidin-4-yl]hepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl]
4- cycloheptylpiperazine-1-carboxylate ##STR00257## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 8.13 (dd, J = 5.0, 2.3 Hz, 1H),
7.64 (dd, J = 5.8, 3.3 Hz, 1H), 7.34-7.55 (m, 1H), 6.20-6.38 (m,
1H), 6.05 (d, J = 11.3 Hz, 1H), 5.78-5.93 (m, 1H), 5.36-5.65 (m,
2H), 4.87-5.13 (m, 2H), 4.53 (br. s., 1H), 3.91-4.19 (m, 3H),
3.53-3.81 (m, 5H), 3.31-3.48 (m, 3H), 3.18 (d, J = 11.5 Hz, 2H),
2.64-2.85 (m, 1H), 2.47- 2.63 (m, 1H), 2.42 (br. s., 1H), 2.34 (br.
s., 1H), 2.20-2.31 (m, 1H), 2.17 (d, J = 6.0 Hz, 1H), 1.84-2.12 (m,
3H), 1.57- 1.81 (m, 8H), 1.55 (s, 2H), 1.51 (s, 4H), 1.39-1.46 (m,
2H), 1.10 (br. s., 1H), 0.94 (dd, J = 6.8, 4.8 Hz, 1H), 0.69-0.90
(m, 7H) 678.9
[(2S,3S,4E,6R)-2-[(2E,4E)-6-[2-[(3R)-3-hydroxypyrrolidin-1-yl]pyrimidin-4-
yl]hepta-2,4-dien-2-yl]-3-methyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4- cycloheptylpiperazine-1-carboxylate ##STR00258## .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.79-1.00 (m, 3 H) 1.28 (t, J =
7.15 Hz, 4 H) 1.42-1.67 (m, 6 H) 1.76 (s, 3 H) 1.78 -1.94 (m, 2
H)2.07 (s, 1 H) 2.20- 2.41 (m, 8 H) 2.52 (ddd, J = 10.04, 6.78,
3.26 Hz, 1 H) 3.40-3.57 (m, 4 H) 3.88 (t, J = 7.28 Hz, 1 H) 4.14
(q, J = 7.28 Hz, 1 H)4.97-5.19 (m, 2 H) 5.38 (dd, J = 14.93, 9.41
Hz, 1 H) 5.44-5.57 (m, 1 H) 6.02-6.21 (m, 2 H) 6.27-6.46 (m, 1 H)
7.08-7.21 (m, 1 H) 8.69-8.76 (m, 2 H) 511.32
[(2S,3S,4E,6R)-3-methyl-12-oxo-2-[(2E,4E)-6-pyrimidin-
2-ylhepta-2,4-dien-2-yl]-1-oxacyclododec-4-en-6-yl] 4-
methylpiperazine-1-carboxylate
Compounds 135-138 were prepared according to the method of Scheme
6.
##STR00259## ##STR00260## ##STR00261##
General Protocol for the Synthesis of Compounds 135-138:
[0610] Step 1: To a solution of potassium tert-butoxide (1.05 g,
8.9 mmol, 1.05 equiv.) under nitrogen in DMF (20 mL, 0.4M) at room
temperature was added 2-methylcyclohexanone MM (1.1 mL, 8.9 mmol,
1.0 equiv.) and the reaction was stirred for 15 hours. The reaction
was cooled to 0.degree. C. and allyl chloroformate (1.1 mL, 10.7
mmol, 1.2 equiv) was added dropwise over 5 min. and stirred for 30
min. The reaction was allowed to warm to room temperature and
quenched into water. The reaction was extracted with 2:1
dichloromethane/hexanes, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The crude material was purified by
silica gel column chromatography (hexanes/diethyl ether as eluant)
to afford the desired product (ally (2-methylcyclohex-1-en-1-yl)
carbonate, NN, 0.7 g, 3.6 mmol, 40%).
[0611] Step 2: To a mixture of
(R)-2-[2-(diphenylphosphino)phenyl]-4-isopropyl-4,5-dihydrooxazole
NN (0.01 g, 0.03 mmol, 0.1 equiv.) and of
tris(dibenzylideneacetone)dipalladium(0) (0.01 g, 0.01 mmol, 0.05
equiv.) was added degassed THF (2.5 mL, 0.01M) under argon. The
reaction was allowed to stir at room temperature for 30 min. Allyl
(2-methylcyclohex-1-en-1-yl) carbonate (0.1 g, 0.25 mmol, 1.0
equiv.) was added and stirred for 8 hr. Then the reaction was
allowed to stand at -20.degree. C. for 16 hr. The reaction was
concentrated in vacuo and the resulting crude material was purified
by silica gel column chromatography (pentane/diethyl ether as
eluent) to afford the desired product
((R)-2-allyl-2-methylcyclohexanone, OO, (0.02 g, 0.12 mmol,
46%).
[0612] Step 3: To a solution of OO (33 mg, 0.22 mmol, 1.0 equiv.)
in dichloromethane (4.0 mL, 0.05M) at 0.degree. C. was added sodium
bicarbonate (0.13 g, 1.2 mmol, 5.6 equiv.) followed by the addition
of peracetic acid (0.17 mL, 0.76 mmol, 30% wt in acetic acid, 3.5
equiv.). The reaction was allowed to warm to room temperature over
4 hours and then stirred at room temperature for an additional 10
hours. The reaction was quenched with sodium bicarbonate and
extracted with dichloromethane. The organic layer was dried over
sodium sulfate and concentrated in vacuo to afford the desired
product (R)-7-allyl-7-methyloxepan-2-one (PP, 0.04 g, 0.22 mmol,
100%) which was advanced crude into the next step.
[0613] Step 4: To a solution of PP (0.04 g, 0.22 mmol, 1.0 equiv.)
in anhydrous methanol (6.0 mL, 0.04M) under nitrogen at room
temperature was added triethylamine (0.15 mL, 5 equiv.). The
reaction was stirred for 8 hours at 90.degree. C. The reaction was
cooled to room temperature and potassium carbonate (6.0 mg, 0.04
mmol, 0.2 equiv.) was added. The reaction was stirred for an
additional 14 hours at room temperature after which time the
reaction was determined to be complete by LCMS or TLC. The reaction
was filtered and concentrated to afford the desired product (QQ,
0.04 g, 0.22 mmol, 100%) which was advanced crude into the next
step.
[0614] Step 5: To a cooled solution of QQ (0.7 g, 3.6 mmol, 1.0
equiv.) and 2,6-lutidine (0.8 mL, 7.2 mmol, 2 equiv.) in
dichloromethane (7 mL, 0.05M) at -78.degree. C. was added dropwise
triethylsilyl trifluoromethansulfonate (0.98 mL, 4.3 mmol, 1.2
equiv.). The reaction was stirred at -78.degree. C. for 1 hour
after which time the reaction was determined to be complete by LCMS
or TLC. The reaction was allowed to warm to room temperature,
quenched with sodium bicarbonate, and extracted with
dichloromethane. The combined organic fractions were washed with
water, brine, dried over sodium sulfate, filtered, and concentrated
in vacuo. The resulting residue was purified by silica gel column
chromatography (hexane/ethyl acetate as eluent) to afford the
desired product (RR, 0.7 g, 2.3 mmol, 63%).
[0615] Step 6: To a cooled solution of RR (0.04 g, 0.13 mmol, 1
equiv.) in THF (2.5 mL, 0.06M) at 0.degree. C. was added hydrogen
peroxide (0.06 mL, 30%, 5 equiv.), followed by a solution of
lithium hydroxide (0.07 g, 0.64 mmol, 5 equiv.) in water (0.5 mL).
The reaction was warmed to room temperature, methanol (8 mL) was
added, and the reaction was stirred for 48 hours at room
temperature. The reaction was quenched with sodium sulfite followed
by saturated citric acid. The mixture was diluted with ethyl
acetate, washed with brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexane/ethyl acetate as eluent) to afford
the desired product (SS, 0.02 g, 0.06 mmol, 47%).
[0616] Step 7: To a solution of acid SS (0.02 g, 0.06 mmol, 1.0
equiv.) in dichloromethane (1.0 mL, 0.06M) at room temperature was
added 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.02 g, 0.09
mmol, 1.5 equiv) and a solution of
(3S,4S,E)-1-iodo-2,4-dimethylhexa-1,5-dien-3-ol (0.023 g, 0.09
mmol, 1.5 equiv.). The reaction was run for 16 hours and determined
to be complete by TLC. The reaction was quenched with water,
diluted with ethyl acetate, washed with brine, dried over sodium
sulfate, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (hexane/ethyl acetate
as eluent) to afford the desired product (TT, 0.02 g, 0.04 mmol,
63%).
[0617] Step 8: To a degassed solution of olefin TT (0.02 g, 0.04
mmol, 1.0 equiv.) and benzoquinone (0.4 mg, 0.004 mmol, 0.1 equiv)
in toluene (10.0 mL, 0.04M) under nitrogen at 20.degree. C. was
added the Hoveyda-Grubbs catalyst (0.006 g, 0.01 mmol, 0.2 equiv.).
The reaction was stirred at 50.degree. C. for 2 hours or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was diluted with ethyl acetate, and the organic layer was washed
with sodium bicarbonate, water, brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (ethyl acetate/hexanes
as eluent) to afford the desired product (UU, 0.01 g, 0.02 mmol,
53%).
[0618] Step 9: To a solution of macrocycle UU (0.01 g, 0.02 mmol,
1.0 equiv.) in dioxane (2 mL, 0.1M) under nitrogen was added
selenium dioxide (0.007 g, 0.06 mmol, 3.0 equiv.) under nitrogen at
room temperature. The reaction was stirred at 85.degree. C. for 20
hours. The reaction was diluted with ethyl acetate, and the organic
layer was washed with sodium bicarbonate, water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (ethyl
acetate/hexanes as eluent) to afford the desired product (VV, 0.007
g, 0.013 mmol, 68%).
[0619] Step 10: To a solution of alcohol VV (1.0 equiv.) in MTBE
(0.1M) at room temperature was added triethylamine (5.0 equiv.),
para-nitrophenylchloroformate (3.0 equiv.), and DMAP (catalytic)
and the reaction was stirred overnight. Once determined to be
complete by TLC or LCMS, the reaction was quenched with water.
Ethyl acetate was added and the organic layer was washed with
water, saturated sodium bicarbonate, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The crude product (XX) was
advanced without purification.
[0620] Step 11: To a solution of carbonate XX (1.0 equiv.) in MTBE
(0.1M) at room temperature was added the corresponding amine (2.0
equiv.). Once determined to be complete by TLC or LCMS, the
reaction was concentrated and the resulting oil was purified by
silica gel column chromatography (hexane/ethyl acetate as eluent)
to afford the desired product (YY).
[0621] Step 12: To a solution of vinyl iodide YY (1.0 equiv.) in
THF (0.1M) at room temperature was added vinyl pinacol boronate
(4.0 equiv.), silver oxide (5.0 equiv.), triphenylarsine (1.2
equiv.), and Pd.sub.2(dba).sub.3 (0.15 equiv.). The reaction was
stirred at room temperature overnight. Once determined to be
complete by TLC or LCMS, the reaction was filtered through
Celite.RTM.. Dichloromethane was added and the organic layer was
washed with water, saturated sodium bicarbonate, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(ZZ).
[0622] Step 13: To a solution of diene ZZ (1.0 equiv.) in
THF:H.sub.2O (0.1M) at 0.degree. C. was added N-methylmorpholine
N-oxide (1.2 equiv.) and osmium tetroxide in t-BuOH (0.1 equiv.).
The reaction was stirred at room temperature overnight. Once
determined to be complete by TLC or LCMS, the reaction was quenched
by addition of aqueous sodium bicarbonate. Ethyl acetate was added
and the organic layer was washed with water, saturated sodium
bicarbonate, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The crude material (AAA) was advanced
without further purification.
[0623] Step 14: To a solution of diol AAA (1.0 equiv.) in acetone:
H.sub.2O (0.1M) at room temperature was added diacetoxyiodobenzene
(1.2 equiv.). The reaction was stirred at room temperature for 40
minutes or until determined to be complete by TLC or LCMS. The
reaction was quenched by addition of sodium thiosulfite and then
sodium bicarbonate. Dichloromethane was added, and the organic
layer was washed with water, saturated sodium bicarbonate, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(BBB).
[0624] Step 15: To a solution of the corresponding sulfone (1.0
equiv.) in THF (0.1M) at -78.degree. C. was added KHMDS (3.0
equiv.) and the reaction was stirred at -78.degree. C. for one
hour. Next, a solution of aldehyde BBB in THF (1.0 equiv.) was
added dropwise at -78.degree. C. The reaction was allowed to warm
gradually to -20.degree. C. and stirred at -20.degree. C. for two
hours. The reaction was quenched with aqueous sodium bicarbonate
and ethyl acetate was added. The organic layer was washed with
water, saturated sodium bicarbonate, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (CCC).
[0625] Step 16: To a solution of silyl ether CCC in methanol (0.1M)
at room temperature was added p-methoxytoluenesulfonic acid (2.5
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate, diluted with ethyl acetate,
washed with water and brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (hexane/ethyl acetate as
eluent) to afford the desired product (135-138), (Table 5).
Exemplified Protocol for the Synthesis of Compound 135
[0626] Steps 1-9 as above.
[0627] Step 10: To a solution of alcohol VV (0.007 g, 0.013 mmol,
1.0 equiv.) in MTBE (1.0 mL, 0.1M) at room temperature was added
triethylamine (0.02 mL, 0.09 mmol, 7.0 equiv.), para-nitrophenyl
chloroformate (0.009 g, 0.05 mmol, 3.5 equiv.), and DMAP (2.0 mg,
0.016 mmol, 1.2 equiv.). The reaction was stirred overnight. Once
determined to be complete by TLC or LCMS, the reaction was quenched
with water. Ethyl acetate was added and the organic layer was
washed with water, saturated sodium bicarbonate, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The crude
product (XX) was advanced without purification.
[0628] Step 11: To a solution of carbonate XX (1.0 equiv.) in MTBE
(1.0 mL, 0.1M) at room temperature was added N-methylpiperazine
(0.007 mL, 0.07 mmol, 5.0 equiv.). Once determined to be complete
by TLC or LCMS, the reaction was concentrated and the resulting oil
was purified by silica gel column chromatography (hexane/ethyl
acetate as eluent) to afford the desired product (YY, 0.008 g,
0.012 mmol, 92%).
[0629] Step 12: To a solution of vinyl iodide YY (0.01 g, 0.015
mmol, 1.0 equiv.) in THF (1.0 mL, 0.01M) at room temperature was
added vinyl pinacol boronate (0.013 mL, 0.08 mmol, 5.0 equiv.),
silver oxide (18.0 mg, 0.08 mmol, 5.0 equiv.), triphenylarsine (5.7
mg, 0.02 mmol, 1.2 equiv.), and Pd.sub.2(dba).sub.3 (3.0 mg, 0.003
mmol, 0.15 equiv.). The reaction was stirred at room temperature
overnight. Once determined to be complete by TLC or LCMS, the
reaction was filtered through Celite.RTM.. Dichloromethane was
added and the organic layer was washed with water and saturated
sodium bicarbonate, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (dichloromethane/methanol as eluent) to
afford the desired product (ZZ, 0.009 g, 0.016 mmol, >95%).
[0630] Step 13: To a solution of diene ZZ (0.009 g, 0.016 mmol, 1.0
equiv.) in THF:H.sub.2O (2.0 mL:0.2 mL, 0.1M) at 0.degree. C. was
added N-methylmorpholine N-oxide (3.2 mg, 0.03 mmol, 1.5 equiv.)
and osmium oxide in t-BuOH (0.05 mL, 0.004 mmol, 0.2 equiv.). The
reaction was stirred at room temperature overnight. Once determined
to be complete by TLC or LCMS, the reaction was quenched by
addition of aqueous sodium bicarbonate. Ethyl acetate was added and
the organic layer was washed with water, saturated sodium
bicarbonate, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The crude material (AAA, 0.008 g, 0.014
mmol, 75%) was advanced without further purification.
[0631] Step 14: To a solution of diol AAA (0.07 g, 0.12 mmol, 1.0
equiv.) in acetone:H20 (5 mL:0.5mL, 0.02M) at room temperature was
added diacetoxyiodobenzene (0.048 g, 0.15 mmol, 1.2 equiv.). The
reaction was stirred at room temperature for 40 minutes or until
determined to be complete by TLC or LCMS. The reaction was quenched
by addition of sodium thiosulfilte and then sodium bicarbonate.
Dichloromethane was added, and the organic layer was washed with
water, saturated sodium bicarbonate, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (BBB, 60 mg, 0.11 mmol,
87%).
[0632] Step 15: To a solution of
(S)-2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl
pyrrolidine-1-carboxylate (0.026 g, 0.07 mmol, 2.5 equiv.) in THF
(2.0 mL, 0.01M) at -78.degree. C. was added KHMDS (0.14 mL, 0.07
mmol, 2.5 equiv.) and the reaction was stirred at -78.degree. C.
for one hour. Next, a solution of aldehyde BBB (0.015 g, 0.03 mmol,
1.0 equiv.) was added dropwise at -78.degree. C. The reaction was
allowed to warm gradually to -20.degree. C. and stirred at
-20.degree. C. for two hours. The reaction was quenched with
aqueous sodium bicarbonate and ethyl acetate was added. The organic
layer was washed with water and saturated sodium bicarbonate, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(CCC, 0.016 mg, 0.023 mmol, 76%).
[0633] Step 16: To a solution of silyl ether CCC (0.016 g, 0.023
mmol, 1 equiv.) in methanol (0.2 mL, 0.1M) at room temperature was
added p-methoxytoluenesulfonic acid (8.0 mg, 0.04 mmol, 1.5
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate, diluted with ethyl acetate,
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel column chromatography (hexane/ethyl acetate as eluent) to
afford the desired product (compound 135, 7 mg, 0.012 mmol, 44%).
.sup.1H NMR (400 MHz, METHANOL-d4) .delta.: 0.89 (d, J=6.78 Hz, 3H)
1.09 (d, J=6.90 Hz, 3H) 1.13-1.31 (m, 5H) 1.35-1.53 (m, 2H)
1.75-1.80 (m, 3H) 1.82-1.94 (m, 5H) 1.94-2.08 (m, 1H) 2.39 (s, 2H)
2.53-2.66 (m, 2H) 2.69 (s, 3H) 2.94 (br. s., 4H) 3.34-3.40 (m, 4H)
3.70 (br. s., 4H) 3.91-4.03 (m, 2H) 4.93-4.99 (m, 1H) 5.07-5.13 (m,
1H) 5.55 (dd, J=15.12, 9.85 Hz, 1H) 5.71 (m, J=9.79 Hz, 2H) 6.13
(d, J=10.67 Hz, 1H) 6.37 (ddd, J=15.12, 10.85, 1.00 Hz, 1H). MS
(ES+)=590.5 [M+H].sup.+.
TABLE-US-00008 TABLE 5 Compounds 135-138 LCMS data Structure,
Compound #, and Chemical Name .sup.1H NMR data (ES+) ##STR00262##
.sup.1H NMR (400 MHz, METHANOL-d4) .delta.: 0.89 (d, J = 6.78 Hz, 3
H) 1.09 (d, J = 6.90 Hz, 3 H) 1.13-1.31 (m, 5 H) 1.35-1.53 (m, 2 H)
1.75- 1.80 (m, 3 H) 1.82-1.94 (m, 5 H) 1.94-2.08 (m, 1 H) 2.39 (s,
2 H) 2.53-2.66 (m, 2 H) 2.69 (s, 3 H) 2.94 (br. s., 4 H) 3.34-3.40
(m, 4 H) 3.70 (br. s., 4 H) 3.91-4.03 (m, 2 H) 4.93- 4.99 (m, 1 H)
5.07-5.13 (m, 1 H) 5.55 (dd, J = 15.12, 9.85 Hz, 1 H) 5.71 (m, J =
9.79 Hz, 2 H) 6.13 (d, J = 10.67 Hz, 1 H) 6.37 (ddd, J = 15.12,
10.85, 1.00 Hz, 1 H) 590.5
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-
(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-
4-en-6-yl] 4-methylpiperazine-1-carboxylate ##STR00263## .sup.1H
NMR (400 MHz, METHANOL-d4) .delta.: 0.89 (d, J = 6.65 Hz, 3 H) 1.10
(d, J = 6.78 Hz, 3 H) 1.13-1.31 (m, 5 H) 1.35-1.53 (m, 2 H) 1.78
(s, 3 H) 1.81-2.08 (m, 6 H) 2.25- 2.46 (m, 2 H) 2.52-2.62 (m, 1 H)
2.64 (s, 3 H) 2.88 (d, J = 2.01 Hz, 4 H) 3.36-3.45 (m, 2 H) 4.00
(d, J = 7.15 Hz, 10 H) 4.95 (d, J = 9.79 Hz, 1 H) 5.10 (d, J =
10.67 Hz, 1 H) 5.55 (dd, J = 15.18, 9.91 Hz, 1 H) 5.70 (m, J = 3.14
Hz, 2 H) 6.11 (d, J = 10.29 Hz, 1 H) 6.38 (dd, J = 15.00, 10.73 Hz,
1 H) 620.5 [(2S,3S,4E,6S,7S)-7-hydroxy-2-[(2E,4E,6R)-7-[(2R)-2-
(hydroxymethyl)pyrrolidine-1-carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-
3,7-dimethyl-12-oxo-1-oxacyclododec-4-en-6-yl]
4-methylpiperazine-1- carboxylate ##STR00264## .sup.1H NMR (400
MHz, METHANOL-d4) .delta.: 0.89 (d, J = 6.65 Hz, 3 H) 1.09 (d, J =
6.90 Hz, 3 H) 1.21 (s, 5 H) 1.31 (s, 8 H) 1.78 (d, J = 0.88 Hz, 3
H) 1.80-2.09 (m, 5 H) 2.32 (s, 3 H) 2.42 (t, J = 5.08 Hz, 6 H)
2.51-2.71 (m, 2 H) 3.41-3.75 (m, 8 H) 3.99 (m, J = 6.71, 4.08 Hz, 2
H) 4.38 (br. s., 1 H) 4.94 (d, J = 9.79 Hz, 1 H) 5.10 (d, J = 10.67
Hz, 1 H) 5.54 (dd, J = 15.18, 9.91 Hz, 1 H) 5.70 (d, J = 9.79 Hz, 2
H) 6.11 (d, J = 10.16 Hz, 1 H) 6.38 (dd, J = 15.06, 10.54 Hz, 1 H)
606.5
[(2S,3S,4E,6S,7S)-7-hydroxy-2-[(2E,4E,6R)-7-[(3R)-3-hydroxypyrrolidine-1-
carbonyl]oxy-6-methylhepta-2,4-dien-2-yl]-3,7-dimethyl-12-oxo-1-oxacyclodo-
dec- 4-en-6-yl] 4-methylpiperazine-1-carboxylate ##STR00265##
.sup.1H NMR (400 MHz, METHANOL-d4) .delta.: 0.77-0.92 (m, 3 H) 1.07
(t, J = 7.65 Hz, 3 H) 1.13-1.31 (m, 5 H) 1.33-1.52 (m, 2 H) 1.72
(s, 3 H) 1.80-1.91 (m, 1 H) 1.92-2.07 (m, 1 H) 2.22-2.46 (m, 4 H)
2.51 (s, 3 H) 2.55-2.65 (m, 1 H) 2.69 (br. s., 4 H) 3.42-3.91 (m, 9
H) 3.99 (d, J = 6.02 Hz, 2 H) 4.94 (d, J = 9.79 Hz, 1 H) 5.01-5.11
(m, 1 H) 5.45-5.59 (m, 1 H) 5.60-5.79 (m, 3 H) 5.97-6.14 (m, 1 H)
6.35 (dd, J = 14.81, 10.79 Hz, 1 H) 7.50-7.66 (m, 3 H) 7.71 (d, J =
7.53 Hz, 2 H) 750.5
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-[(3S)-3--
(1-phenyltetrazol-
5-yl)oxypyrrolidine-1-carbonyl]oxyhepta-2,4-dien-2-yl]-12-oxo-1-oxacyclodo-
dec-4-en-6-yl] 4-methylpiperazine-1-carboxylate
Compounds 139-142 were prepared by the method of Scheme 7.
##STR00266## ##STR00267## ##STR00268##
General Protocol for the Synthesis of Compounds 139-142
[0634] Step 1: To a solution of methyl 4-oxopentanoate DDD (10.0 g,
76.8 mmol, 1.0 equiv.) and allyltrimethylsilane (13.4 mL, 84.5
mmol, 1.1 equiv.) under nitrogen in THF (13.4 mL, 6M) at room
temperature was added TBAF (1.0 g, 3.8 mmol, 0.05 equiv.) and
4.ANG. molecular sieves (0.2 equiv. wt). The reaction was stirred
at reflux for 36 hours. The reaction was filtered and concentrated
in vacuo. The crude material was purified by silica gel column
chromatography (hexanes/ethyl acetate as eluant) to afford the
desired product (5-allyl-5-methyldihydrofuran-2(3H)-one) (EEE, 5.8
g, 21.7 mmol, 28%).
[0635] Step 2: To a cooled solution of N,O-dimethylhydroxylamine
hydrochloride (1.7 g, 17.8 mmol, 5.0 equiv.) in THF (20.0 mL, 0.9M)
under nitrogen at 0.degree. C. was added trimethylaluminum (7.1 mL,
14.3 mmol, 4.0 equiv.). The reaction was stirred at room
temperature for 30 min. A solution of
5-allyl-5-methyldihydrofuran-2(3H)-one, EEE, (0.5 g, 3.6 mmol, 1.0
equiv.) in THF (5.0 mL) was added at 0.degree. C. and the reaction
was stirred for 2 hours. The reaction was poured onto a cooled
mixture of ethyl acetate and saturated potassium tartrate and
stirred for 15 mins. The organic layer was separated and the
aqueous layer was extracted with diethyl ether. The combined
organic fractions were dried over sodium sulfate, filtered, and
concentrated in vacuo to afford the desired product
(4-hydroxy-N-methoxy-N,4-dimethylhept-6-enamide, FFF) which was
advanced crude into the next step.
[0636] Step 3: To a solution of
4-hydroxy-N-methoxy-N,4-dimethylhept-6-enamide FFF (1.0 equiv) in
DMF (20.0 mL, 0.9M) at room temperature was added 1H-imidazole (1.2
g, 17.8 mmol, 5.0 equiv.) and chlorotriethylsilane (2.4 mL, 14.3
mmol, 4.0 equiv.). The reaction was stirred at room temperature for
12 hours. The reaction was diluted with brine and extracted with
diethyl ether. The combined organic fractions were dried over
sodium sulfate, filtered, and concentrated in vacuo. The resulting
residue was purified by silica gel column chromatography
(hexanes/ethyl acetate as eluent) to afford the desired product
(N-methoxy-N,4-dimethyl-4-((triethylsilyl)oxy)hept-6-enamide (GGG,
0.56 g, 1.8 mmol, 50%).
[0637] Step 4: To a solution of amide GGG (0.25 g, 0.79 mmol, 1.0
equiv.) in THF (6.0 mL, 0.13M) under nitrogen at -78.degree. C. was
added DIBAL-H (1.3 mL, 1.3 mmol, 1.6 equiv.) and stirred for one
hour. The reaction was quenched with aqueous hydrochloric acid (1M)
and stirred for an additional 15 min. The reaction was extracted
with ethyl acetate and the combined organic fractions were
concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexanes/ethyl acetate as eluent) to afford
the desired product (4-methyl-4-((triethylsilyl)oxy)hept-6-enal,
HHH, 0.19 g, 0.74 mmol, 93%).
[0638] Step 5: To a solution of
(S)-3-acetyl-4-benzyloxazolidin-2-one HHH (0.15 g, 0.68 mmol, 1
equiv.) in dichloromethane (3.0 mL, 0.2M) under nitrogen at
-78.degree. C. was added
dibutyl(((trifluoromethyl)sulfonyl)oxy)borane (0.75 mL, 0.75 mmol,
1M toluene, 1.1 equiv.), followed by diisopropylethylamine (0.15
mL, 0.89 mmol, 1.3 equiv.). The reaction was sucessively stirred at
-78.degree. C. for 15 min., at 0.degree. C. for 1 hour, and then at
-78.degree. C. for 30 min. To the cooled reation mixture was added
dropwise 4-methyl-4-((triethylsilyl)oxy)hept-6-enal (0.17 g, 0.68
mmol, 1.0 equiv.) followed by stirring at room temperature for 2
hours. The reaction was quenched with ammonia chloride and
extracted with dichloromethane. The combined organic fractions were
dried over sodium sulfate and concentrated in vacuo. The residue
was purified by silica gel column chromatography (hexanes/ethyl
acetate as eluent) to afford the desired diastereomeric products as
a separable mixture (III-A and III-B). The stereochemistry of each
diastereoisomer was assigned according to their NOE data and cross
peak between H4 and H8.
##STR00269##
[0639] .sup.1H NMR (CHLOROFORM-d) .delta.: 7.09-7.28 (m, 2H), 5.74
(dd, J=17.7, 9.2 Hz, 1H), 4.96 (d, J=11.8 Hz, 1H), 4.54-4.65 (m,
1H), 3.99-4.14 (m, 1H), 3.15-3.27 (m, 1H), 3.05-3.14 (m, 1H),
3.00-3.04 (m, 1H), 2.88-2.98 (m, 1H), 2.63-2.71 (m, 1H), 2.15 (d,
J=6.3 Hz, 1H), 1.52-1.59 (m, 1H), 1.20-1.49 (m, 4H), 1.10-1.16 (m,
1H), 0.68-0.91 (m, 7H), 0.47-0.56 (m, 2H).
[0640] .sup.13C NMR (CHLOROFORM-d) .delta.: 171.2, 153.5, 153.4,
135.4, 135.3, 135.2, 135.1, 135.0, 135.0, 129.4, 129.0, 129.0,
128.9, 127.4, 127.4, 127.3, 117.2, 117.2, 117.1, 117.1, 77.3, 76.4,
75.0, 74.9, 68.5, 66.3, 66.2, 66.2, 66.1, 55.2, 55.2, 55.1, 55.1,
47.3, 46.9, 38.0, 37.9, 37.8, 33.8, 31.2, 31.1, 31.0, 29.8, 27.8,
27.7, 27.6, 27.6, 26.6, 26.5, 26.5, 25.8, 25.5, 25.5, 25.3, 19.2,
19.1, 19.0, 18.9, 14.1, 14.0, 14.0, 13.9, 13.8, 13.8, 7.2, 6.9.
##STR00270##
[0641] .sup.1H NMR (CHLOROFORM-d) .delta.: 7.27-7.37 (m, 6H), 7.22
(d, J=6.8 Hz, 4H), 5.78-5.87 (m, 1H), 5.03-5.09 (m, 3H), 4.66-4.74
(m, 2H), 4.06-4.25 (m, 6H), 3.28-3.34 (m, 2H), 3.05-3.12 (m, 3H),
2.79-2.83 (m, 1H), 2.57 (s, 2H), 2.20-2.29 (m, 3H), 1.59-1.70 (m,
4H), 1.45-1.54 (m, 2H), 1.31-1.41 (m, 1H), 1.20-1.29 (m, 6H),
0.93-1.00 (m, 15H), 0.57-0.64 (m, 9H).
[0642] .sup.13C NMR (CHLOROFORM-d) .delta.: 172.8, 172.8, 171.1,
170.3, 153.7, 153.5, 135.3, 135.1, 135.0, 135.0, 129.4, 129.0,
129.0, 128.9, 127.4, 127.4, 117.2, 117.2, 76.8, 75.0, 75.0, 68.6,
68.5, 66.3, 66.1, 60.4, 55.1, 55.1, 55.0, 53.5, 47.3, 47.0, 42.9,
42.8, 38.0, 38.0, 37.8, 37.8, 31.1, 31.1, 30.7, 27.8, 27.6, 26.6,
25.5, 23.8, 21.0, 19.1, 14.2, 14.0, 13.9, 13.7, 7.2, 6.9, 6.8,
6.6.
[0643] Similar protocols were used for III-A and III-B
[0644] Step 6: To a solution of alcohol III-A (1.0 equiv) in DMF
(0.09M) at room temperature was added 1H-imidazole (5.0 equiv.) and
tert-butylchlorodimethylsilane (2.5 equiv.). The reaction was
stirred under nitrogen at room temperature for 3 hours. The
reaction was diluted with brine and extracted with ethyl acetate.
The combined organic fractions were dried over sodium sulfate,
filtered, and concentrated in vacuo. The resulting residue was
purified by silica gel column chromatography (hexanes/ethyl acetate
as eluent) to afford the desired product (JJJ-A).
[0645] Step 7: To a cooled solution JJJ-A (1.0 equiv.) in THF
(0.9M) at 0.degree. C. was added hydrogen peroxide (7.6 equiv.),
followed by a solution of lithium hydroxide (8.0 equiv.) in water
(0.8M). The reaction was stirred at 0.degree. C. for 1 hour and
then at room temperature for 3 hours. The reaction was quenched
with sodium thiosulfate. The mixture was extracted with ethyl
acetate, acidified to pH 3, and extracted with ethyl acetate. The
organic layer was dried over sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexanes/ethyl acetate as eluent) to afford
the desired acid (KKK-A). Step 8: To a solution of acid KKK-A (1.0
equiv.) and freshly prepared
(3S,4S,E)-1-iodo-2,4-dimethylhexa-1,5-dien-3-ol (1.4 equiv.) (For
protocols related to the synthesis of
(3S,4S,E)-1-iodo-2,4-dimethylhexa-1,5-dien-3-ol, see: Kumar, V. P.;
Chandrasekhar, S. Org. Lett. 2013, 15, 3610-3613) in
dichloromethane (0.08M) at room temperature was added
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (1.3 equiv) and DMAP
(catalytic). The reaction was stirred at room temperature for 16
hours and determined to be complete by TLC. The reaction was
concentrated in vacuo and the residue was purified by silica gel
column chromatography (hexane/ethyl acetate) to afford the desired
ester (LLL-A).
[0646] Step 9: To a degassed solution of olefin LLL-A (1.0 equiv.)
and benzoquinone (0.05 equiv) in toluene (0.01M) under nitrogen at
20.degree. C. was added the Hoveyda-Grubbs catalyst 0.1 equiv.).
The reaction was stirred at 50.degree. C. for 2 hours or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was diluted with ethyl acetate, and the organic layer was washed
with sodium bicarbonate, water, brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (ethyl acetate/hexanes
as eluent) to afford the desired product (MMM-A).
[0647] Step 10: To solution of solution of macrocycle MMM-A (1.0
equiv.) in dioxane (0.1M) under nitrogen was added selenium dioxide
(4.0 equiv.) under nitrogen at room temperature. The reaction was
stirred at 85.degree. C. for 20 hours. The reaction was diluted
with ethyl acetate, and the organic layer was washed with sodium
bicarbonate, water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (ethyl acetate/hexanes as eluent) to afford
the desired diastereoisomer products: a separable mixture (NNN-A)
and (000-A). The stereochemistry of each diastereoisomer was
assigned according to their COSY, HMBC, HMQC and NOESY data.
##STR00271##
[0648] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm -0.03-0.03
(m, 4H) 0.49-0.63 (m, 5H) 0.80-0.94 (m, 15H) 1.16-1.23 (m, 3H)
1.25-1.45 (m,3H) 1.39-1.50 (m, 6H) 1.63-1.86 (m, 3H) 2.06 (s, 1H)
2.22-2.48 (m, 2H) 3.73 (dd, J=8.16, 3.89 Hz, 1H) 3.92 (d, J=10.29
Hz, 1H) 3.88-3.96 (m, 1H) 5.06 (d, J=10.79 Hz, 1H) 5.33 (dd,
J=15.06, 9.79 Hz, 1H) 5.49 (dd, J=15.06, 9.79 Hz, 1H) 6.40 (d,
J=1.00 Hz, 1H) 6.37-6.44 (m,1H) 7.20 (s, 3H).
[0649] .sup.13C NMR (100 MHz, CHLOROFORM-d) .delta. ppm -4.9, -4.8,
6.8, 7.1, 16.5, 18.1, 19.3, 22.9, 25.8, 29.0, 36.9, 40.4, 40.6,
70.2, 76.7, 78.2, 79.3, 80.8, 83.8, 128.9, 138.4, 143.8, 168.7.
##STR00272##
[0650] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm -0.03-0.02
(m, 6H) 0.47-0.68 (m, 6H) 0.78-0.95 (m, 21H) 1.15-1.35 (m, 6H)
1.37-1.58 (m, 7H) 1.66-1.80 (m, 3H) 2.24-2.48 (m, 3H) 2.56 (d,
J=10.79 Hz, 1H) 3.49 (t, J=10.16 Hz, 1H) 3.64-3.80 (m, 1H) 5.05 (d,
J=10.54 Hz, 1H) 5.29 (dd, J=15.31, 9.79 Hz, 1H) 5.53 (dd, J=15.31,
9.54 Hz, 1H) 6.38 (d, J=1.00 Hz, 1H) 7.19 (s, 3H).
[0651] .sup.13C NMR (101 MHz, CHLOROFORM-d) .delta. ppm -4.8, -4.7,
6.8, 7.1, 16.6, 18.1, 19.2, 24.6, 25.7, 25.8, 30.7, 37.8, 40.6,
41.0, 70.8, 77.2, 77.8, 77.9, 80.5, 83.6, 130.9, 135.6, 143.9,
168.6. NNN-B and OOO-B were isolated using similar procedures
starting from III-B
##STR00273##
[0652] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm -0.06-0.04
(m, 6H) -0.02-0.02 (m, 6H) 0.48-0.66 (m, 6H) 0.77-0.94 (m, 20H)
1.16-1.25 (m, 4H) 1.26-1.44 (m, 2H) 1.50 (s, 2H) 1.56-1.72 (m, 2H)
1.74-1.80 (m, 3H) 2.04 (s, 1H) 2.29-2.49 (m, 3H) 2.40-2.50 (m, 1H)
3.80-4.02 (m, 1H) 4.24 (td, J=6.34, 2.64 Hz, 1H) 5.16 (d, J=10.54
Hz, 1H) 5.31-5.41 (m, 2H) 6.39 (d, J=1.00 Hz, 1H) 7.20 (s, 1H).
[0653] .sup.13C NMR (100 MHz, CHLOROFORM-d) .delta. ppm -5.2, -4.8,
6.7, 7.2, 16.5, 18.0, 19.2, 23.1, 25.8, 26.5, 32.1, 40.6, 40.9,
68.5, 77.2, 78.0, 79. 0, 80.0 , 83.7, 129.5, 137.9, 143.8,
170.1.
##STR00274##
[0654] .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta. ppm -0.03-0.04
(m, 6H) 0.49-0.70 (m, 6H) 0.78-0.98 (m, 21H) 1.13-1.35 (m, 7H) 1.51
(s, 3H), 1.55-1.67 (m, 1H) 1.69-1.88 (m, 4H) 2.31-2.52 (m, 3H) 2.60
(d, J=11.04 Hz, 1H) 3.45 (t, J=10.29 Hz, 1H) 4.16-4.32 (m, 1H)
5.05-5.25, (m, 2H) 5.57 (dd, J=15.31, 9.79 Hz, 1H) 6.40 (d, J=1.00
Hz, 1H) 7.22 (s, 1H).
[0655] .sup.13C NMR (100 MHz, CHLOROFORM-d) .delta. ppm -4.90, 6.8,
6.9, 6.9, 7.1, 7.2, 16.4, 17.8, 19.1, 24.3, 25.6, 25.8, 28.1, 29.7,
31.6, 40.4, 41.2, 67.8, 77.2, 77.7, 78.0, 79.9, 83.6, 131.6, 134.6,
143.9, 170.0.
[0656] Step 11: To a solution of NNN-A (1.0 equiv.) in THF (0.1M)
at room temperature was added the corresponding
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (2.0 equiv.),
monosilver(I) monosilver(III) monooxide (5.0 equiv.),
triphenylarsine (1.2 equiv.), and tetrakis(triphenylphosphine)
palladium (0.15 equiv.). The reaction mixture was heated at
60.degree. C. for 30 minutes, or until the reaction was determined
to be complete by LCMS or TLC. Upon completion, the reaction was
cooled down to room temperature, the mixture was then filtered
through Celite.RTM., washed with dichloromethane and concentrated
in vacuo. The crude material was purified by silica gel
chromatography (dichloromethane/methanol as eluent) to afford the
desired product (PPP-A)
[0657] Step 12: To a solution of alcohol PPP-A (1.0 equiv.) in
dichloromethane (0.1M) at room temperature was added DMAP (0.5
equiv.) followed by 4-nitrophenyl chloroformate (2.0 equiv.). The
reaction was stirred at room temperature for three hours. Next, the
corresponding amine (3.0 equiv.) was added at room temperature.
After stirring for one hour, the reaction was quenched with water
and diluted with dichloromethane. The organic layer was washed with
1N sodium hydroxide solution, and the organic layer was
concentrated. The resulting oil was purified by silica gel column
chromatography (hexanes/ethyl acetate as eluant) to afford the
desired product (QQQ-A).
[0658] Step 13: To a solution of silyl ether QQQ-A (1 equiv.) in
methanol (0.1M) at room temperature was added
p-methoxytoluenesulfonic acid (1.5 equiv.). The reaction was
stirred for 3 hours, or until the reaction was determined to be
complete by LCMS or TLC. The reaction was quenched with sodium
bicarbonate, diluted with ethyl acetate, washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(RRR-A).
[0659] Other diastereoisomers (OOO-A, NNN-B and OOO-B) were
subjected to the procedures to afford compounds 139-142.
Exemplified Protocol for the Synthesis of Compound 140
[0660] Steps 1-5 as above.
[0661] Step 6: To a solution of alcohol III-A (0.14 g, 0.3 mmol,
1.0 equiv) in DMF (3.2 mL, 0.09M) at room temperature was added
1H-imidazole (0.10 g, 1.5 mmol, 5.0 equiv.) and
tert-butylchlorodimethylsilane (0.11 g, 0.75 mmol, 2.5 equiv.). The
reaction was stirred under nitrogen at room temperature for 3
hours. The reaction was diluted with brine and extracted with ethyl
acetate. The combined organic fractions were dried over sodium
sulfate, filtered, and concentrated in vacuo. The resulting residue
was purified by silica gel column chromatography (hexanes/ethyl
acetate as eluent) to afford the desired product (JJJ-A, 0.16 g,
0.27 mmol, 92%).
[0662] Step 7: To a cooled solution of JJJ-A (0.16 g, 0.27 mmol,
1.0 equiv.) in THF (0.9M) at 0.degree. C. was added hydrogen
peroxide (0.25 mL, 2.3 mmol, 30%, 7.6 equiv.), followed by a
solution of lithium hydroxide (0.06 g, 2.4 mmol, 8.0 equiv.) in
water (3.0 mL, 0.8M). The reaction was stirred at 0.degree. C. for
1 hour and then at room temperature for 3 hours. The reaction was
quenched with sodium thiosulfate. The mixture was extracted with
ethyl acetate, acidified to pH 3, and extracted with ethyl acetate.
The organic layer was dried over sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (hexanes/ethyl acetate as eluent) to afford
the desired acid (KKK-A, 0.10 g, 0.24 mmol, 80%).
[0663] Step 8: To a solution of acid KKK-A (0.10 g, 0.24 mmol, 1.0
equiv.) and (3S,4S,E)-1-iodo-2,4-dimethylhexa-1,5-dien-3-ol (0.16
g, 0.45 mmol, 1.9 equiv.) in dichloromethane (3.0 mL, 0.08M) at
room temperature was added
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (0.06 g, 0.31 mmol,
1.3 equiv) and one crystal of DMAP. The reaction was stirred at
room temperature for 16 hours and determined to be complete by TLC.
The reaction was concentrated in vacuo and the residue was purified
by silica gel column chromatography (hexane/ethyl acetate) to
afford the desired ester (LLL-A, 0.17 g, 0.25 mmol, >95%).
[0664] Step 9: To a degassed solution of olefin LLL-A (41.0 mg,
0.06 mmol, 1.0 equiv.) and benzoquinone (0.4 mg, 0.003 mmol, 0.05
equiv) in toluene (6.0 mL, 0.01M) under nitrogen at 20.degree. C.
was added the Hoveyda-Grubbs catalyst (4.0 mg, 0.006 mmol, 0.1
equiv.). The reaction was stirred at 50.degree. C. for 2 hours or
until the reaction was determined to be complete by LCMS or TLC.
The reaction was diluted with ethyl acetate, and the organic layer
was washed with sodium bicarbonate, water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The crude
macrocycle (MMM-A) was carried on to the next step without further
purification.
[0665] Step 10: To a solution of macrocycle MMM-A (1.0 equiv.) in
dioxane (2 mL, 0.1M) under nitrogen was added selenium dioxide
(30.0 mg, 0.25 mmol, 4.0 equiv.) under nitrogen at room
temperature. The reaction was stirred at 85.degree. C. for 20
hours. The reaction was diluted with ethyl acetate, and the organic
layer was washed with sodium bicarbonate, water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (ethyl
acetate/hexanes as eluent) to afford the desired separable
diastereoisomer products as (NNN-A, 5.5 mg, 0.008 mmol, 13%) and
(OOO-A, 6.4 mg, 0.010 mmol, 16%).
[0666] Step 11: To a solution of NNN-A (10.0 mg, 0.015 mmol, 1.0
equiv.) in THF (1.0 mL, 0.01M) at room temperature was added
(R,E)-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)but-3-en-1--
yl pyrrolidine-1-carboxylate (18.0 mg, 0.06 mmol, 3.8 equiv.),
monosilver(I) monosilver(III) monooxide (14.2 mg, 0.06 mmol, 4.0
equiv.), triphenylarsine (5.6 mg, 0.02 mmol, 1.2 equiv.), and
tetrakis(triphenylphosphine) palladium (2.1 mg, 0.002 mmol, 0.15
equiv.). The reaction mixture was heated to 60.degree. C. for 30
minutes, or until the reaction was determined to be complete by
LCMS or TLC. Upon completion, the reaction was cooled down to room
temperature, the mixture was then filtered through Celite.RTM.,
washed with dichloromethane and concentrated in vacuo. The crude
material was purified by silica gel chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(PPP-A, 5.3 mg, 0.006 mmol, 80%).
[0667] Step 12: To a solution of alcohol PPP-A (5.0 mg, 0.007 mmol,
1.0 equiv.) in dichloromethane (0.5 mL, 0.01M) at room temperature
was added N,N-dimethylaminopyridine (0.4 mg, 0.003 mmol, 0.5
equiv.) followed by 4-nitrophenyl chloroformate (5.0 mg, 0.02 mmol,
3.5 equiv.). The reaction was stirred at room temperature for three
hours. Next, N-methyl piperazine (0.004 mL, 0.03 mmol, 4.5 equiv.)
was added at room temperature. After stirring for one hour, the
reaction was quenched with water and diluted with dichloromethane.
The organic layer was washed with 1N sodium hydroxide solution, and
the organic layer was concentrated. The resulting crude carbamate
(QQQ-A) was used in the nest step without further purification.
[0668] Step 13: To a solution of carbamate QQQ-A (1.0 equiv.) in
methanol (0.1M) at room temperature was added
p-methoxytoluenesulfonic acid (2.7 mg, 0.014 mmol, 2.0 equiv.). The
reaction was stirred for 3 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate, diluted with ethyl acetate, washed with
water, brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (dichloromethane/methanol as eluent) to
afford the desired product (compound 140, 2.7 mg, 0.005 mmol, 63%).
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.00 (s, 1H) 0.06 (s,
1H) 0.65 (s, 1H) 0.74-0.96 (m, 3H) 0.98-1.18 (m, 3H) 1.18-1.45 (m,
5H) 1.61 (d, J=7.28 Hz, 1H) 1.68-1.81 (m, 2H) 1.84 (br. s., 3H)
2.05 (d, J=11.80 Hz, 2H) 2.37 (s, 4H) 2.44 (d, J=14.56 Hz, 2H)
2.50-2.77 (m, 5H) 3.13 (d, J=18.32 Hz, 1H) 3.24 (d, J=6.27 Hz, 1H)
3.26-3.42 (m, 3H) 3.31 (br. s., 1H) 3.37 (br. s., 2H) 3.56 (br. s.,
2H) 3.48-3.64 (m, 3H) 3.64 (br. s., 1H) 3.70 (d, J=2.26 Hz, 1H)
3.82-4.03 (m, 2H) 5.16 (dd, J=10.04, 8.03 Hz, 2H) 5.29 (s, 1H) 5.39
(dd, J=14.93, 10.16 Hz, 1H), 5.52-5.75 (m, 2H) 6.09 (d, J=11.29 Hz,
1H) 6.17-6.40 (m, 1H) 6.98 (s, 1H) 7.25 (s, 5H) 7.51 (s, 1H).
TABLE-US-00009 TABLE 6 Compounds 139-142 LCMS data Structure,
Compound#, and Chemical Name .sup.1H NMR data (ES+) ##STR00275##
.sup.1H NMR (400 MHz, CHLOROFORM- d) .delta.: 0.00 (s, 1 H)
0.72-0.91 (m, 4 H) 0.92-1.09 (m, 4 H) 1.10-1.30 (m, 4 H) 1.34-1.56
(m, 3 H) 1.56-1.69 (m, 4 H) 1.72-1.81 (m, 4 H) 1.84 (br. s., 1 H)
2.22-2.40 (m, 5 H) 2.40-2.57 (m, 7 H) 3.05 (s, 2 H) 3.09 (s, 1 H)
3.19 (br. s., 1 H) 3.22-3.28 (m, 2 H) 3.31 (br. s., 2 H) 3.49 (br.
s., 4 H) 3.64 (s, 1 H) 3.78- 4.01 (m, 2 H) 4.31 (br. s., 1 H) 4.89-
5.11 (m, 2 H) 5.35 (dd, J = 15.31,10.04 Hz, 1 H) 5.51 (dd, J =
14.81, 9.79 Hz, 1 H) 5.62 (dd, J = 15.06, 7.53 Hz, 1 H) 6.03 (d, J
= 10.79 Hz, 1 H) 6.20 (dd, J = 14.93, 10.92 Hz, 1 H) 7.19 (s, 2H)
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-
-
(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-e-
n- 6-yl] 4-methylpiperazine-1-carboxylate ##STR00276## .sup.1H NMR
(400 MHz, CHLOROFORM- d) .delta.: 0.00 (s, 1 H) 0.06 (s, 1 H) 0.65
(s, 1 H) 0.74-0.96 (m, 3 H) 0.98-1.18 (m, 3 H) 1.18-1.45 (m, 5H)
1.61 (d, J = 7.28 Hz, 1 H) 1.68-1.81 (m, 2 H) 1.84 (br. s., 3 H)
2.05 (d, J = 11.80 Hz, 2 H) 2.37 (s, 4 H) 2.44 (d, J = 14.56 Hz, 2
H) 2.50-2.77 (m, 5 H) 3.13 (d, J = 18.32 Hz, 1 H) 3.24 (d, J = 6.27
Hz, 1 H) 3.26-3.42 (m, 3 H) 3.31 (br. s., 1 H) 3.37 (br. s., 2 H)
3.56 (br. s., 2 H) 3.48- 3.64 (m, 3H) 3.64 (br. s., 1 H) 3.70 (d, J
= 2.26 Hz, 1 H) 3.82-4.03 (m, 2 H) 5.16 (dd, J = 10.04, 8.03 Hz, 2
H) 5.29 (s, 1 H) 5.39 (dd, J = 14.93, 10.16 Hz, 1 H), 5.52-5.75 (m,
2 H) 6.09 (d, J = 11.29 Hz, 1 H) 6.17-6.40 (m, 1 H) 6.98 (s, 1 H)
7.25 (s, 5 H) 7.51 (s, 1 H)
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-
-
(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-e-
n- 6-yl] 4-methylpiperazine-1-carboxylate ##STR00277## .sup.1H NMR
(400 MHz, METHANOL- d4) .delta.: 0.87 (d, J = 6.78 Hz, 3 H) 1.16-
1.25 (m, 5 H) 1.45 (d, J = 7.03 Hz, 5 H) 1.77 (s, 3 H) 1.81-1.91
(m, 1 H) 1.92-2.10 (m, 1 H) 2.26-2.38 (m, 2 H) 2.45 (s, 3 H)
2.55-2.65 (m, 5 H) 3.43- 3.85 (m, 5 H) 4.94 (d, J = 9.79 Hz, 1 H)
5.09 (d, J = 10.67 Hz, 1 H) 5.53 (dd, J = 15.06, 9.91 Hz, 1 H) 5.73
(m, J = 9.66 Hz, 1 H) 6.00 (dd, J = 15.18, 7.53 Hz, 1 H) 6.13 (d, J
= 10.67 Hz, 1 H) 6.39 (dd, J = 15.06, 10.79 Hz, 1 H) 7.26-7.30 (m,
1 H) 7.35 (d, J = 7.91 Hz, 1 H) 7.79 (td, J = 7.72, 1.76 Hz, 1 H)
8.46 (d, J = 4.52 Hz, 1 H) 562.3
[(2S,3S,4E,6S,7S)-7-hydroxy-3,7-dimethyl-12-oxo-2-
[(2E,4E,6S)-6-pyridin-2-ylhepta-2,4-dien-2-yl]-1-
oxacyclododec-4-en-6-yl] 4-methylpiperazine-1-carboxylate
##STR00278## .sup.1H NMR (400 MHz, CHLOROFORM- d) .delta.: 0.00 (s,
1 H) 0.65-0.90 (m, 3 H) 0.91-1.07 (m, 3 H) 1.09-1.24 (m, 3 H)
1.28-1.45 (m, 2 H) 1.46-1.74 (m, 6 H) 1.78 (br. s., 4 H) 2.03 (br.
s., 5 H) 2.35 (s, 4 H) 2.40-2.55 (m, 6 H) 2.82 (s, 1 H) 2.89 (s, 1
H) 3.25 (d, J = 5.27 Hz, 2 H) 3.31 (br. s., 2 H) 3.54 (br. s., 3 H)
3.89 (qd, J = 10.71, 6.78 Hz, 2 H) 4.30 (br. s., 1 H) 4.94 (dd, J =
18.07, 10.04 Hz, 2 H) 5.48 (dd, J = 15.31, 10.04 Hz, 1 H) 5.55-5.83
(m, 2 H) 5.55-5.72 (m, 1 H) 6.02 (d, J = 11.29 Hz, 1 H) 6.20 (dd, J
= 14.31, 11.04 Hz, 1 H) 6.93 (s, 1 H) 7.19 (s, 6 H) 7.45 (s, 1 H)
[(2S,3S,4E,6S,7S,10S)-7,10-dihydroxy-3,7-dimethyl-2-[(2E,4E,6R)-6-methyl-7-
-
(pyrrolidine-1-carbonyloxy)hepta-2,4-dien-2-yl]-12-oxo-1-oxacyclododec-4-e-
n- 6-yl] 4-methylpiperazine-1-carboxylate
Synthesis of Sulfone Intermediates for Preparation of Urea
Compounds
##STR00279##
[0669] General Protocol for the Synthesis of Sulfone Urea Side
Chain
[0670] Step 1: To a solution of R(-)-3-bromo-2-methyl-1-propanol
SSS (4.0 g, 26.1 mmol, 1.0 equiv.) in DMF (20.0 mL, 1.3M), was
added sodium azide (5.1 g, 78.4 mmol, 3.0 equiv.). The mixture was
warmed up to 100.degree. C. and stirred at 100.degree. C. for 4
hours, or until the reaction was determined to be complete by LCMS
or TLC. After cooling down to room temperature, the mixture was
filtered to remove solid, and washed with diethyl ether. The
filtrate was washed with water and brine. After drying over sodium
sulfate, filtration and evaporation of the solvent, the crude azido
derivative (TTT, 2.4 g, 20.8 mmol, 78%) was used in the next
step.
[0671] Step 2: To the mixture of (S)-3-azido-2-methylpropan-1-ol
TTT (2.4 g, 20.8 mmol, 1.0 equiv.) and BOC-anhydride (6.8 g, 31.3
mmol, 1.5 equiv.) in THF (100 mL, 0.2M) under nitrogen was added
Pd-C (2.2 g, 2.1 mmol, 0.1 equiv.). The reaction was purged and
then placed under hydrogen atmosphere, and stirred for 16 hours, or
until the reaction was determined to be complete by LCMS or TLC.
The reaction atmosphere was substituted with nitrogen and the
precipitate was removed via filtration through Celite.RTM.. The
Celite.RTM. was washed with MeOH and the solvent was removed under
reduced pressure. The crude material was purified by silica gel
column chromatography (hexane/ethyl acetate) to give the desired
product (UUU, 2.6 g, 13.8 mmol, 66%).
[0672] Step 3: To the solution of Boc-protected amine UUU (2.6 g,
13.8 mmol, 1.0 equiv.), 1-phenyl-1H-tetrazole-5-thiol (2.6 g, 14.5
mmol, 1.05 equiv.) and triphenylphosphine (3.8 g, 14.5 mmol, 1.05
equiv.) in THF (100 mL, 0.1M) at 0.degree. C., DIAD (3.2 ml, 16.5
mmol, 1.2 equiv.) was added dropwise. The reaction mixture
maintained at 0.degree. C. and stirred for 2 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
mixture was then diluted with ethyl acetate, and washed with water
and brine. After drying over sodium sulfate, filtration and
evaporation of solvent, the crude material was purified via silica
gel (hexane/ethyl acetate) to give the desired product (VVV, 4.3 g,
12.2 mmol, 89%).
[0673] Step 4: To the solution of the tetrazole VVV (0.4 g, 1.1
mmol, 1.0 equiv.) in dichloromethane (7.0 mL, 0.14M) at 0.degree.
C., trifluoroacetic acid (3.5 mL, 45.4 mmol, 40.0 equiv.) was
added. The reaction mixture was allowed to warm up to 23.degree. C.
and stirred for 1 hour, or until the reaction was determined to be
complete by LCMS or TLC. The solvent was removed under reduced
pressure, and the reaction was diluted with ethyl acetate. The
organic layer was washed with sodium bicarbonate and brine. After
drying over sodium sulfate, filtration and evaporation of the
solvent, the crude amine (WWW, 0.3 g, 1.0 mmol, 90%) was used in
the next step.
[0674] Step 5: To the solution of the amine WWW (1.0 equiv.) in
dichloromethane (0.1M) at 0.degree. C., diisopropylethylamine (4.0
equiv.) and the corresponding carbamic chloride (2.0 equiv.) were
added and stirred for 2 hours at the same temperature. Once
completion of reaction was confirmed by LCMS or TLC, the solution
was diluted with dichloromethane. The organic layer was washed with
ammonium chloride, sodium bicarbonate, and brine. After drying with
sodium sulfate, filtration and evaporation of the solvent, the
crude was purified with silica gel (dichloromethane/methanol) to
give the desired urea (XXX).
[0675] Step 6: To a solution of urea XXX (1.0 equiv.) in ethanol
(0.1M) at 0.degree. C. was added dropwise a premixed yellow
solution of ammonium molybdate tetrahydrate (0.3 equiv.) in 33%
hydrogen peroxide (10 equiv.). The reaction mixture was allowed to
warm up to room temperature and stirred for 4 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
mixture was diluted in ethyl acetate then sodium thiosulfate was
added at 0.degree. C. and stirred for 20 minutes. The organic layer
was then washed with water, brine, and dried over sodium sulfate.
After evaporation of the solvent, the residue was purified by
silica gel column chromatography (dichloromethane/methanol) to give
the desired sulfone (YYY).
Exemplified Protocol for the Synthesis of
(S)--N--(2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl)pyrrolidi-
ne-1-carboxamide.
[0676] Steps 1-4 as above.
[0677] Step 5: To the solution of the amine WWW (0.1 g, 0.4 mmol, 1
equiv.) in dichloromethane (4.4 mL, 0.1M) at 0.degree. C.,
diisopropylethylamine (0.3 mL, 1.7 mmol, 4 equiv.) and
pyrrolidine-1-carbonyl chloride (0.1 mL, 0.8 mmol, 2.0 equiv.) were
added and stirred for an additional 2 hours at the same
temperature. Once completion of reaction was confirmed by LCMS or
TLC, the solution was diluted with dichloromethane. The organic
layer was washed with ammonium chloride, sodium bicarbonate, and
brine. After drying with sodium sulfate, filtration and evaporation
of the solvent, the crude was purified by silica gel column
chromatography (dichloromethane/methanol) to give the desired urea
(XXX, 0.13 g, 0.4 mmol, 91%).
[0678] Step 6: To a solution of product urea XXX (0.1 g, 0.4 mmol,
1.0 equiv.) in ethanol (3.0 mL, 0.1M) at 0.degree. C. was added
dropwise a premixed yellow solution of ammonium molybdate
tetrahydrate (0.1 g, 0.12 mmol, 0.3 equiv.) in 33% hydrogen
peroxide (0.4 mL, 4.0 mmol, 10 equiv.). The reaction mixture was
allowed to warm up to room temperature and stirred for 4 hours, or
until the reaction was determined to be complete by LCMS or TLC.
The reaction mixture was diluted in ethyl acetate and sodium
thiosulfate was added at 0.degree. C. Stirring was continued for an
additional 20 minutes. The organic layer was then washed with
water, brine, and dried over sodium sulfate. After evaporation of
the solvent, the residue was purified by silica gel column
chromatography (dichloromethane/methanol) to give the desired
sulfone,
(S)--N--(2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl)pyrrolidi-
ne-l-carboxamide (0.1 g, 0.28 mmol, 68%). [0679] Compounds 143 and
144 were prepared using sulfone YYY according to Scheme 9.
##STR00280##
[0679] General Protocol for the Synthesis of Urea Compounds
143-144
[0680] Step 1: To a solution of sulfone YYY (2.5 equiv.) in THF
(0.02M) under nitrogen at -78.degree. C. was added KHMDS (2.5
equiv.) dropwise and the reaction was stirred for 20 minutes. Then
aldehyde L (1.0 equiv.) in THF (0.5 M) was added dropwise. The
reaction was stirred at -78.degree. C. for 90 minutes and then
allowed to warm to -20.degree. C. for 1 hour. The reaction was
quenched with ammonium chloride and diluted with ethyl acetate. The
organic layer was washed with water, brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The resulting oil was
purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(ZZZ).
[0681] Step 2: To a solution of product ZZZ (1.0 equiv.) in
methanol (0.1M) was added p-toluenesulfonic acid (3.0 equiv.) at
room temperature. The reaction was stirred for 2 hours, or until
the reaction was determined to be complete by LCMS or TLC. The
reaction was quenched with sodium bicarbonate. The mixture was
diluted with EtOAc. The aqueous layer was extracted with EtOAc. The
combined organic layers were washed with brine, dried over sodium
sulfate and concentrated in vacuo. The crude residue was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (compounds 143-144).
Exemplified Protocol for the Synthesis of Urea Compound 143
[0682] Step 1: To a solution of
(S)--N--(2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl)pyrrolidi-
ne-1-carboxamide YYY (43.7 mg, 0.12 mmol, 2.5 equiv.) in THF (3.0
mL, 0.02M) under nitrogen at -78.degree. C. was added dropwise
KHMDS (0.23 mL, 0.12 mmol, 2.5 equiv.) and the reaction was stirred
for 20 minutes. Then aldehyde L (30.0 mg, 0.05 mmol, 1.0 equiv.) in
THF was added dropwise. The reaction was stirred at -78.degree. C.
for 90 minutes and then allowed to warm to -20.degree. C. for 1
hour. The reaction was quenched with ammonium chloride and diluted
with ethyl acetate. The organic layer was washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(ZZZ, 23.3 mg, 0.03 mmol, 63%).
[0683] Step 2: To a solution of product ZZZ (23.3 mg, 0.03 mmol,
1.0 equiv.) in methanol (3.0 mL, 0.1M) was added p-toluenesulfonic
acid (16.6 mg, 0.09 mmol, 3.0 equiv.) at room temperature. The
reaction was stirred for 2 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate. The mixture was diluted with EtOAc. The
aqueous layer was extracted with EtOAc. The combined organic layers
were washed with brine, dried over sodium sulfate and concentrated
in vacuo. The crude residue was purified by silica gel column
chromatography (dichloromethane/methanol as eluent) to afford the
desired product (compound 143, 15.6 mg, 0.02 mmol, 78%). .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta.: 0.91 (d, J=6.78 Hz, 3H)
0.99-1.08 (d, J=6.65 Hz, 3H) 1.27 (d, J=5.27 Hz, 3H) 1.30-1.61 (m,
17H) 1.90 (s, 3H) 1.60-2.06 (m, 3H) 2.45-2.67 (m, 4H) 2.70-2.84 (m,
5H) 2.84-2.97 (m, 1H) 2.95-3.13 (m, 1H) 3.22-3.36 (m, 4H) 3.61-3.71
(m, 4H) 3.73-3.81 (m, 1H) 5.02 (d, J=9.41 Hz, 1H) 5.16 (d, J=10.67
Hz, 1H) 5.56-5.75 (m, 3H) 6.06-6.12 (d, J=10.92 Hz, 1H) 6.27 (dd,
J=15.12, 11.11 Hz, 1H). MS (ES+)=687.6 [M+H].sup.+. [0684] Compound
145 was prepared by the method of Scheme 10.
##STR00281## ##STR00282##
[0684] Exemplified Protocol for the Synthesis of Urea Compound
145
[0685] Step 1: To a solution of
(S)--N--(2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl)pyrrolidi-
ne-1-carboxamide (34.1 mg, 0.09 mmol, 2.5 equiv.) in THF (2.0 mL,
0.02M) under nitrogen at -78.degree. C. was added KHMDS (0.18 mL,
0.09 mmol, 2.5 equiv.) dropwise and the reaction was stirred for 20
minutes. Then aldehyde E (20.0 mg, 0.04 mmol, 1.0 equiv.) in THF
(0.5 M) was added dropwise. The reaction was stirred at -78.degree.
C. for 90 minutes and then allowed to warm to -20.degree. C. for 1
hour. The reaction was quenched with ammonium chloride and diluted
with ethyl acetate. The organic layer was washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product
(AAAA, 15.8 mg, 0.02 mmol, 62%).
[0686] Step 2: To a solution of urea AAAA (25.2 mg, 0.04 mmol, 1.0
equiv.) in methanol (3.0 mL, 0.01M) was added potassium carbonate
(14.8 mg, 0.11 mmol, 3.0 equiv.) and stirred at room temperature.
After 3 hours, or until the reaction was determined to be complete
by LCMS or TLC, the reaction was quenched with ammonium chloride at
0.degree. C. The mixture was then diluted with ethyl acetate. The
organic layer was washed with water, brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The resulting oil was
purified by silica gel column chromatography (hexane/ethyl acetate
as eluent) to afford the desired secondary alcohol (BBBB, 26.0 mg,
0.04 mmol, >95%).
[0687] Step 3: To a solution of alcohol BBBB (26.0 mg, 0.04 mmol,
1.0 equiv.) in dichloromethane (2.0 mL, 0.1M) was added
diisopropylethylamine (0.05 mL, 0.27 mmol, 7.0 equiv.) and DMAP
(1.4 mg, 0.01 mmol, 0.3 equiv.) at 0.degree. C. Then a solution of
4-nitrophenyl carbonochloridate (31.5 mg, 0.16 mmol, 4.0 equiv.) in
dichloromethane (0.1M) was added slowly. The reaction was warmed up
to room temperature and stirred for 3 hours, or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
quenched with sodium bicarbonate and diluted with dichloromethane.
The aqueous layer was extracted with dichloromethane. The combined
organic layers were washed with brine, dried over sodium sulfate
and concentrated in vacuo. The crude protected carbonate (CCCC) was
used in the next step without further purification.
[0688] Step 4: To a solution of carbonate CCCC (1.0 equiv.) in THF
(0.1M) at room temperature was added N-methylpiperazine (0.04
mL,0.4 mmol, 10.0 equiv.) at room temperature. After stirring for
one hour, or until the reaction was determined to be complete by
LCMS or TLC, the reaction was quenched with water and diluted with
ethyl acetate, washed with 1N sodium hydroxide solution, and the
organic layer was concentrated. The resulting oil was purified by
silica gel column chromatography (dichloromethane/methanol as
eluant) to afford the desired product (DDDD, 15.1 mg, 0.02 mmol,
49%).
[0689] Step 5: To a solution of carbamate DDDD (15.2 mg, 0.02 mmol,
1.0 equiv.) in methanol (2.0 mL, 0.01M) at room temperature was
added p-methoxytoluenesulfonic acid (11.0 mg, 0.06 mmol, 3.0
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(compound 145, 4.5 mg, 0.008 mmol, 39%). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.73 -0.89 (m, 3H) 0.90-1.04 (m, 3H)
1.08-1.33 (m, 6H) 1.40-1.58 (m, 2H) 1.59-1.71 (m, 4H) 1.75-1.90 (m,
4H) 2.24 (s, 3H) 2.31 (br. s., 3H) 2.34-2.57 (m, 3H) 2.90-3.16 (m,
1H) 3.16-3.30 (m, 4H) 3.37-3.50 (m, 4H) 3.53-3.77 (m, 1H) 4.13 (t,
J=5.77 Hz, 1H) 4.95 (d, J=9.54 Hz, 1H) 5.08 (d, J=10.79 Hz, 1H)
5.49-5.67 (m, 2H) 6.02 (d, J=11.04 Hz, 1H) 6.10-6.34 (m, 1H). MS
(ES+)=605.5 [MAH].sup.+. [0690] Compound 146 was prepared by the
method of Scheme 11
##STR00283## ##STR00284##
[0690] Exemplified Protocol for the Synthesis of Urea Compound
146
[0691] Step 1: To a solution of
(S)-5-((3-((tert-butyldimethylsily)oxy)-2-methylpropyl)sulfonyl)-1-phenyl-
-1H-tetrazole (29.3 mg, 0.07 mmol, 2.0 equiv.) in THF (2.0 mL,
0.02M) under nitrogen at -78.degree. C. was added KHMDS (0.15 mL,
0.07 mmol, 2.0 equiv.) dropwise and the reaction was stirred for 20
minutes. Then aldehyde L (24.0 mg, 0.04 mmol, 1.0 equiv.) in THF
(0.5 M) was added dropwise. The reaction was stirred at -78.degree.
C. for 90 minutes and then allowed to warm to -20.degree. C. over 1
hour. The reaction was quenched with ammonium chloride and diluted
with ethyl acetate. The organic layer was washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired diene (EEEE,
15.6 mg, 0.02 mmol, 51.5%).
[0692] Step 2: To a solution of diene EEEE (15.6 mg, 0.02 mmol, 1.0
equiv.) in methanol (2.0 mL, 0.02M) was added p-toluenesulfonic
acid (2.3 mg, 0.01 mmol, 0.6 equiv.) at room temperature. The
reaction was stirred for 2 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate. The mixture was diluted with ethyl
acetate. The aqueous layer was extracted with ethyl acetate. The
combined organic layers were washed with brine, dried over sodium
sulfate and concentrated in vacuo. The crude residue was purified
by silica gel column chromatography (hexanes/ethyl acetate as
eluent) to afford the desired alcohol (FFFF, 9.0 mg, 0.01 mmol,
60%).
[0693] Step 3: To a solution of alcohol FFFF (7.5 mg, 0.01 mmol,
1.0 equiv.) in dichloromethane (1.0 mL, 0.01M) was added DMAP (1.6
mg, 0.01 mmol, 1.3 equiv.) and tosyl chloride (2.0 mg, 0.01 mmol,
1.0 equiv.) at 0.degree. C. The reaction was warmed up to room
temperature and the reaction was stirred for 24 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was then quenched with water and washed with brine. After drying
over sodium sulfate, filtration and evaporation of solvent, the
crude tosylate (1 equiv.) was then dissolved in DMF (1.0 mL, 0.01M)
and sodium azide (2.7 mg, 0.04 mmol, 4.0 equiv.) was added. The
reaction was warmed to 70.degree. C. and stirred for 4 hours, or
until the reaction was determined to be complete by LCMS or TLC.
Upon completion, the excess of solvent was removed and the crude
material was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired azide
(GGGG, 6.0 mg, 0.01 mmol, 96%).
[0694] Step 4: To a solution of dichloromethane (1.0 mL, 0.01M)
containing azide GGGG (7.5 mg, 0.01 mmol, 1.0 equiv.) was added a
trimethylphosphine (0.02 mL, 0.02 mmol, 2.0 equiv.) toluene
solution (1M) at room temperature. The reaction was stirred for 3
hours, or until the reaction was determined to be complete by LCMS
or TLC. Paraformaldehyde (1.5 mg, 0.05 mmol, 5.0 equiv.) was added
at room temperature and the mixture was stirred for 5 hours, or
until the reaction was determined to be complete by LCMS or TLC.
Methanol was added (1 mL/1.0 equiv. of GGGG) and the reaction was
cooled to 0.degree. C. Sodium borohydride (2.0 mg, 0.05 mmol, 5.0
equiv.) was added and the reaction was stirred at 0.degree. C. for
1 hour or until the reaction was determined to be complete by LCMS
or TLC. The reaction was then quenched with sodium bicarbonate,
extracted with dichloromethane, and dried over sodium sulfate.
After filtration and evaporation, the crude amine (HHHH, 7.4 mg,
0.01 mmol, >95%) was used in the next step without further
purification.
[0695] Step 5: To a solution of amine HHHH (7.0 mg, 0.01 mmol, 1
equiv.) in dichloromethane (1.0 mL, 0.01M) was added triethylamine
(0.005 mL, 0.04 mmol, 4.0 equiv.) at room temperature. The reaction
mixture was then cooled down to 0.degree. C. and then
pyrrolidine-1-carbonyl chloride (2.6 mg, 0.02 mmol, 2.0 equiv.) was
added slowly. After warming up to room temperature, the reaction
was stirred for 3 hours, or until the reaction was determined to be
complete by LCMS or TLC. Upon completion of the reaction, excess of
solvent was removed and crude material was then purified using
silica gel chromatography (dichloromethane/methanol as eluent) to
afford the desired urea (IIII, 2.5 mg, 0.003 mmol, 32%).
[0696] Step 6: A solution of urea IIII (2.5 mg, 0.003 mmol, 1.0
equiv.) in methanol (2.0 mL, 0.01M) at room temperature was added
p-methoxytoluenesulfonic acid (1.2 mg, 0.006 mmol, 2.0 equiv.). The
reaction was stirred for 3 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate and diluted with ethyl acetate. The organic
layer was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (compound 146, 1.8 mg, 0.003
mmol, 84%). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.84-1.08
(m, 10H) 1.25-1.41 (m, 9H) 1.44-1.68 (m, 8H) 1.70-1.85 (m, 5H)
1.88-2.05 (m, 3H) 2.42-2.65 (m, 6H) 2.80-2.95 (m, 6H) 3.08-3.18 (m,
2H) 3.54-3.73 (m, 5H) 3.78 (br. s., 1H) 4.94 (d, J=9.66 Hz, 1H)
5.04 (d, J=10.67 Hz, 1H) 5.54-5.63 (m, 2H) 5.66-5.77 (m, 1H) 6.08
(d, J=11.04 Hz, 1H) 6.31 (dd, J=14.87, 10.98 Hz, 1H), MS
(ES+)=701.4 [M+H].sup.+.
TABLE-US-00010 TABLE 7 Compounds 143-146 LCMS data Structure,
Compound #, and Chemical Name .sup.1H NMR data (ES+) ##STR00285##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.91 (d, J = 6.78 Hz,
3 H) 0.99-1.08 (d, J = 6.65 Hz, 3 H) 1.27 (d, J = 5.27 Hz, 3 H)
1.30-1.61 (m, 17 H) 1.90 (s, 3 H) 1.60-2.06 (m, 3 H) 2.45-2.67 (m,
4 H) 2.70-2.84 (m, 5 H) 2.84- 2.97 (m, 1 H) 2.95-3.13 (m, 1 H)
3.22-3.36 (m, 4 H) 3.61-3.71 (m, 4 H) 3.73-3.81 (m, 1 H) 5.02 (d, J
= 9.41 Hz, 1 H) 5.16 (d, J = 10.67 Hz, 1 H) 5.56-5.75 (m, 3 H)
6.06-6.12 (d, J = 10.92 Hz, 1 H) 6.27 (dd, J = 15.12, 11.11 Hz, 1
H) 687.6 ##STR00286## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.71-0.92 (m, 4 H) 0.93-1.13 (m, 4 H) 1.15- 1.25 (m, 5 H) 1.27 (br.
s., 1 H) 1.30- 1.54 (m, 12 H) 1.56-1.67 (m, 7 H) 1.69-1.76 (m, 2 H)
1.80-1.95 (m, 3 H) 2.36-2.57 (m, 8 H) 2.98 (ddd, J = 13.18, 8.03,
4.89 Hz, 1 H) 3.09 (s, 1 H) 3.14-3.33 (m, 3 H) 3.33-3.49 (m, 6 H)
3.57 (d, J = 10.29 Hz, 1 H) 3.61-3.82 (m, 1 H) 3.86-4.13 (m, 1 H)
4.49 (br. s., 1 H) 4.94 (d, J = 9.54 Hz, 2 H) 5.08 (d, J = 10.54
Hz, 1 H) 5.46-5.68 (m, 3 H) 6.02 (d, J = 11.04 Hz, 1 H) 6.20 (dd, J
= 15.18, 10.67 Hz, 1 H) 717.7 ##STR00287## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.73-0.89 (m, 3 H) 0.90-1.04 (m, 3 H) 1.08-
1.33 (m, 6 H) 1.40-1.58 (m, 2 H) 1.59-1.71 (m, 4 H) 1.75-1.90 (m, 4
H) 2.24 (s, 3 H) 2.31 (br. s., 3 H) 2.34-2.57 (m, 3 H) 2.90-3.16
(m, 1 H) 3.16-3.30 (m, 4 H) 3.37-3.50 (m, 4 H) 3.53-3.77 (m, 1 H)
4.13 (t, J = 5.77 Hz, 1 H) 4.95 (d, J = 9.54 Hz, 1 H) 5.08 (d, J =
10.79 Hz, 1 H) 5.49- 5.67 (m, 2 H) 6.02 (d, J = 11.04 Hz, 1 H)
6.10-6.34 (m, 1 H) 605.5 ##STR00288## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.84-1.08 (m, 10 H) 1.25-1.41 (m, 9 H) 1.44-
1.68 (m, 8 H) 1.70-1.85 (m, 5 H) 1.88-2.05 (m, 3 H) 2.42-2.65 (m, 6
H) 2.80-2.95 (m, 6 H) 3.08-3.18 (m, 2 H) 3.54-3.73 (m, 5 H) 3.78
(br. s., 1 H) 4.94 (d, J = 9.66 Hz, 1 H) 5.04 (d, J = 10.67 Hz, 1
H) 5.54-5.63 (m, 2 H) 5.66-5.77 (m, 1 H) 6.08 (d, J = 11.04 Hz, 1
H) 6.31 (dd, J = 14.87, 10.98 Hz, 1 H) 701.4
Compound 147 was prepared as shown in Scheme 12.
##STR00289##
Protocol for the Synthesis of Compound 147
[0697] Step 1: To a solution of compound diene EEEE (22.0 mg, 0.03
mmol, 1.0 equiv.) in methanol (2.0 mL, 0.01M) was added
p-toluenesulfonic acid (15.5 mg, 0.08 mmol, 3.0 equiv.) at room
temperature. The reaction was stirred for 2 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate. The mixture was diluted with
EtOAc. The aqueous layer was extracted with EtOAc. The combined
organic layers were washed with brine, dried over sodium sulfate
and concentrated in vacuo. The crude residue was purified by silica
gel chromatography (hexanes/ethyl acetate as eluent) to afford the
desired diol (JJJJ, 5.0 mg, 0.008 mmol, 32%).
[0698] Step 2: To a solution of diene JJJJ (6.0 mg, 0.01 mmol, 1
equiv.) in dichloromethane (1.0 mL, 0.01M) was added DMAP (1.6 mg,
1.3 equiv.) and tosyl chloride (2.0 mg, 0.01 mmol, 1.0 equiv.) at
0.degree. C. The reaction was warmed up to room temperature and the
reaction was stirred for 24 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was then
quenched with water, and washed with brine. After drying over
sodium sulfate, filtration and evaporation of solvent, the crude
tosylate (1 equiv.) was then dissolved in DMF (1.0 mL, 0.01M) and
sodium azide (2.6 mg, 0.04 mmol, 4.0 equiv.) was added. The
reaction was warmed to 70.degree. C. and stirred for 4 hours, or
until the reaction was determined to be complete by LCMS or TLC.
Upon completion, the excess of solvent was removed and the crude
material was purified by silica gel chromatography
(dichloromethane/methanol as eluent) to afford the desired azide
(KKKK, 6.0 mg, 0.01 mmol, 96%).
[0699] Step 3: To a solution of product KKKK (5.0 mg, 0.008 mmol,
1.0 equiv.) in water/tert-butanol/dichloromethane (0.1M, 1/2/1,
0.25/0.5/0.25 mL) was added ethynylcyclopropane (2.2 mg, 0.03 mmol,
4.0 equiv.), copper (II) sulfate (2.0 mg, 0.01 mmol, 1.5 equiv.),
and sodium
(R)-5-((S)-1,2-dihydroxyethyl)-4-hydroxy-2-oxo-2,5-dihydrofuran-3-olate
(3.2 mg, 0.02 mmol, 2.0 equiv.). The reaction was stirred at room
temperature for 7 hours, or until the reaction was determined to be
complete by LCMS or TLC. Upon completion, the excess of solvent was
removed and the crude material was purified by silica gel
chromatography (dichloromethane/methanol as eluent) to afford the
desired triazole (Compound 147, 2.5 mg, 0.004 mmol, 45%). .sup.1H
NMR (400 MHz, METHANOL-d4) 0.78-0.98 (m, 6H) 0.99-1.06 (m, 3H)
1.19-1.28 (m, 4H) 1.28-1.43 (m, 2H) 1.50-1.62 (m, 14H) 1.63-1.75
(m, 6H) 1.85-1.99 (m, 2H) 2.41-2.68 (m, 7H) 2.73-2.89 (m, 1H)
3.39-3.62 (m, 4H) 3.75 (br. s., 2H) 4.07-4.26 (m, 2H) 5.01 (d,
J=9.54 Hz, 1H) 5.13 (d, J=10.67 Hz, 1H) 5.55-5.74 (m, 3H) 6.01-6.07
(m, 1H) 6.10-6.20 (m, 1H), MS (ES+)=683.5 [M+H].sup.+.
Compound 148 was prepared by the method of Scheme 13.
##STR00290## ##STR00291##
Protocol for the Synthesis of Compound 148
[0700] Step 1: To a solution of
(S)-5-((3-((tert-butyldimethylsilyl)oxy)-2-methylpropyl)sulfonyl)-1-pheny-
l-1H-tetrazole (104.0 mg, 0.26 mmol, 2.5 equiv.) in THF (10.0 mL,
0.01M) under nitrogen at -78.degree. C. was added KHMDS (0.52 mL,
0.26 mmol, 2.5 equiv.) dropwise and the reaction was stirred for 20
minutes. Then aldehyde E (58.0 mg, 0.1 mmol, 1.0 equiv.) in THF
(0.5 M) was added dropwise. The reaction was stirred at -78.degree.
C. for 90 minutes and then allowed to warm to -20.degree. C. for 1
hour. The reaction was quenched with ammonium chloride and diluted
with ethyl acetate. The organic layer was washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired diene (LLLL,
45.0 mg, 0.06 mmol, 59%).
[0701] Step 2: To a solution of diene LLLL (40.0 mg, 0.05 mmol, 1.0
equiv.) in methanol (4.0 mL, 0.01M) was added potassium carbonate
(19.1 mg, 0.14 mmol, 2.5 equiv.) and the reaction was stirred at
room temperature. After 3 hours, or until the reaction was
determined to be complete by LCMS or TLC, the reaction was quenched
with ammonium chloride at 0.degree. C. The mixture was then diluted
with ethyl acetate. The organic layer was washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired secondary
alcohol (MMMM, 40.0 mg, 0.06 mmol, >95%).
[0702] Step 3: To a solution of alcohol MMMM (40.0 mg, 0.06 mmol,
1.0 equiv.) in dichloromethane (6.0 mL, 0.01M) was added
triethylamine (0.06 mL, 0.4 mmol, 7.0 equiv.), and DMAP (2.1 mg,
0.02 mmol, 0.3 equiv.) at 0.degree. C. Then a solution of
4-nitrophenyl carbonochloridate (47.2 mg, 0.23 mmol, 4.0 equiv.) in
dichloromethane (0.1M) was added slowly. The reaction was warmed up
to room temperature and stirred for 3 hours, or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
quenched with sodium bicarbonate and diluted with dichloromethane.
The aqueous layer was extracted with dichloromethane. The combined
organic layers were washed with brine, dried over sodium sulfate
and concentrated in vacuo. The crude protected carbonate (NNNN) was
used in the next step without further purification.
[0703] Step 4: To a solution of carbamate NNNN (1.0 equiv.) in THF
(6.0 mL, 0.01M) at room temperature was added N-methyl piperazine
(0.07 mL, 0.58 mmol, 10.0 equiv.). After stirring for one hour, or
until the reaction was determined to be complete by LCMS or TLC,
the reaction was quenched with water and diluted with EtOAc. The
organic layer was washed with 1N sodium hydroxide solution and the
organic layer was concentrated. The resulting oil was purified by
silica gel column chromatography (dichloromethane/methanol as
eluant) to afford the desired carbamate (0000, 37.0 mg, 0.04 mmol,
78%).
[0704] Step 5: To a solution of carbamate OOOO (37.0 mg, 0.05 mmol,
1.0 equiv.) in methanol (4.0 mL, 0.01M) at room temperature was
added p-methoxytoluenesulfonic acid (19.1 mg, 0.1 mmol, 2.2
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired alcohol
(PPPP, 18.5 mg, 0.04 mmol, 80%).
[0705] Step 6: To a solution of alcohol PPPP (45.0 mg, 0.09 mmol,
1.0 equiv.) in dichloromethane (1.0 mL, 0.08M) was added
triethylamine (0.15 mL, 0.9 mmol, 10.0 equiv.), and DMAP (2.2 mg,
0.02 mmol, 0.2 equiv.) at 0.degree. C. Then a solution of
4-nitrophenyl carbonochloridate (26.7 mg, 0.13 mmol, 1.5 equiv.) in
dichloromethane (0.1M) was added slowly. The reaction was warmed up
to room temperature and stirred for 3 hours, or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
quenched with sodium bicarbonate and diluted with dichloromethane.
The aqueous layer was extracted with dichloromethane. The combined
organic layers were washed with brine, dried over sodium sulfate
and concentrated in vacuo. The crude carbonate (QQQQ) was used in
the next step without further purification.
[0706] Step 7: To a solution of carbonate QQQQ (1.0 equiv.) in
dichloromethane (1.0 mL, 0.03M) at room temperature was added the
require amine (5.0 equiv.) at room temperature. After stirring for
one hour, or until the reaction was determined to be complete by
LCMS or TLC, the mixture was concentrated. The resulting oil was
purified by silica gel column chromatography
(dichloromethane/methanol as eluant) to afford the corresponding
carbamate and desired carbonate as a minor by product (compound
148, 1.0 mg, 0.002 mmol, 4.7%). .sup.1H NMR (400 MHz, METHANOL-d4)
.delta.: 0.91 (d, J=6.65 Hz, 3H) 1.09 (d, J=6.78 Hz, 3H) 1.11-1.19
(m, 1H) 1.23 (s, 3H) 1.30-1.46 (m, 3H) 1.50-1.69 (m, 2H) 1.77 (s,
3H) 2.40 (s, 3H) 2.46-2.71 (m, 7H) 3.41-3.70 (m, 4H) 3.75 (s, 3H)
3.78-3.84 (m, 1H) 3.92-4.09 (m, 2H) 4.96 (d, J=9.54 Hz, 1H) 5.07
(d, J=10.67 Hz, 1H) 5.53-5.81 (m, 3H) 6.11 (d, J=10.54 Hz, 1H) 6.38
(dd, J=15.00, 10.85 Hz, 1H), MS (ES+)=566.5 [M+H].sup.+.
Compound 149 was prepared according to Scheme 14.
##STR00292##
Protocol for the Synthesis of Compound 149
[0707] Step 1: To a solution of (R)-methyl
3-hydroxy-2-methylpropanoate RRRR (5.0 g, 42.3 mmol, 1.0 equiv.) in
dichloromethane (125 mL, 0.3 M) was added imidazole (4.3 g, 63.5
mmol, 1.5 equiv.) followed by TBS-Cl (6.2 g, 63.5 mmol, 1.2 equiv.)
and the reaction was stirred at room temperature for 6 hours, or
until the reaction was determined to be complete by LCMS or TLC.
The reaction was then quenched with water. The organic layer was
then washed with brine and dried over sodium sulfate. After
filtration, the solvent was removed in vacuo and the residue was
purified by silica gel column chromatography (hexane/ethyl acetate
as eluant) to afford the desired product to give the desired
protected ester (SSSS, 7.0 g, 30.1 mmol, 71%).
[0708] Step 2: To a stirred solution of TBS protected ester SSSS
(7.0 g, 30.1 mmol, 1.0 equiv.) in dry dichloromethane (80 mL, 0.2M)
at -10.degree. C. was added DIBAL-H (1.0 M solution in toluene,
75.3 mL, 75.3 mmol 2.5 equiv.) under nitrogen. The reaction mixture
was stirred at 0.degree. C. for 30 minutes and allowed to warm to
room temperature and further stirred for an additional 2 hours, or
until the reaction was determined to be complete by LCMS or TLC.
After the excess DIBAL-H was decomposed with an excess of methanol,
the mixture was poured into a sodium potassium tartrate solution
(10 g in 100 mL water) with vigorous stirring until the layers were
separated. The aqueous layer was extracted with diethyl ether and
the combined organic extracts were washed with brine, dried over
sodium sulfate and concentrated. The crude product was purified by
silica gel column chromatography (hexane/ethyl acetate as eluant)
to afford the desired product (TTTT, 4.8 g, 23.5 mmol, 78%).
[0709] Step 3: To a solution of TBS protected alcohol TTTT (1.0 g,
4.9 mmol, 1.0 equiv.) in dichloromethane (20 mL, 0.1M) was added
sodium bicarbonate (0.8 g, 9.8 mmol, 2.0 equiv.) at room
temperature. Then a solution of DMP (3.1 g, 7.3 mmol, 1.5 equiv.)
in dichloromethane (5 mL) was added dropwise. The reaction was
stirred for 2 hours, or until the reaction was determined to be
complete by LCMS or TLC. The solvent was removed and the crude
aldehyde was then quickly purified on a silica plug (hexane/ethyl
acetate as eluant). The aldehyde was then dissolved in benzene (25
mL, 0.1M) and methyl (triphenylphosphoranylidene)acetate (1.6 g,
4.9 mmol, 1.0 equiv.) was added to the reaction at room temperature
and the reaction was stirred for 10 hours, or until the reaction
was determined to be complete by LCMS or TLC. After concentration,
the residue was purified by silica gel column chromatography
(hexane/ethyl acetate as eluant) to afford the desired ester (UUUU,
400 mg, 1.6 mmol, 32%).
[0710] Step 4: To a solution of the ester UUUU (200 mg, 0.77 mmol,
1.0 equiv.) in methanol (7 mL, 0.1M) was added palladium/carbon
(10%, 82 mg, 0.77 mmol Pd, 0.1 equiv.) under a nitrogen atmosphere.
The reaction was then placed under a hydrogen atmosphere and
stirred at room temperature for 2 hours, or until the reaction was
determined to be complete by LCMS or TLC. Upon completion, the
hydrogen atmosphere was replaced by a nitrogen atmosphere and then
the palladium/carbon was filtered off on a Celite.RTM. pad and the
excess solvent was removed to give the desired product (VVVV, 101
mg, 0.691 mmol, 89%).
[0711] Step 5: To a solution of ester VVVV (100 mg, 0.68 mmol, 1.0
equiv.) in THF (4 mL, 0.1M) was added triphenylphosphine (206 mg,
0.79 mmol, 1.15 equiv.) followed by 1-phenyl-1H-tetrazole-5-thiol
(134 mg, 0.75 mmol, 1.1 equiv.). The reaction was degassed with
nitrogen and a solution of DIAD (180 mg, 0.89 mmol, 1.3 equiv.) in
THF (2 mL) was added slowly. The reaction was then stirred at room
temperature for 1 hour, or until the reaction was determined to be
complete by LCMS or TLC. The solvent was removed. The crude residue
was then purified by silica gel column chromatography (hexane/ethyl
acetate as eluant) to afford the desired product (WWWW, 110 mg,
0.36 mmol, 53%).
[0712] Step 6: To a solution of sulfide WWWW (25 mg, 0.082 mmol,
1.0 equiv.) in ethanol (1 mL, 0.1M) at 0.degree. C. was added
dropwise a premixed yellow solution of ammonium molybdate
tetrahydrate (20 mg, 0.016 mmol, 0.2 equiv.) in hydrogen peroxide
(33% in water, 0.084 mL, 0.82 mmol, 10.0 equiv.). The reaction
mixture was allowed to warm up to room temperature and stirred for
4 hours, or until the reaction was determined to be complete by
LCMS or TLC. The reaction mixture was diluted in ethyl acetate then
sodium thiosulfate was added at 0.degree. C. and the reaction was
stirred for 20 minutes. The organic layer was then washed with
water, brine, and dried over sodium sulfate. After evaporation of
the solvent, the residue was purified by silica gel column
chromatography (hexane/ethyl acetate) to give the desired sulfone
(XXXX, 17 mg, 0.050 mmol, 62%).
[0713] Step 7: To a solution of the sulfone XXXX (23 mg, 0.069
mmol, 1.5 equiv.) in THF (1 mL, 0.02M) under nitrogen at
-78.degree. C. was added KHMDS (0.5 M in toluene, 0.19 mL, 0.092
mmol, 2.0 equiv.) dropwise and the reaction was stirred for 20
minutes. Then aldehyde L (30 mg, 0.046 mmol, 1.0 equiv.) (see
Scheme 2) in THF (1 mL) was added dropwise. The reaction was
stirred at -78.degree. C. for 90 minutes and then allowed to warm
to -20.degree. C. for 1 hour. The reaction was quenched with
ammonium chloride and diluted with ethyl acetate. The organic layer
was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (hexane/ethyl acetate as
eluent) to afford the desired product (YYYY, 12 mg, 0.016 mmol,
34%).
[0714] Step 8: To a solution of ester YYYY (11 mg, 0.014 mmol, 1.0
equiv.) in methanol (1.5 mL, 0.01M) at room temperature was added
p-methoxytoluenesulfonic acid (8.3 mg, 0.043 mmol, 3.0 equiv.). The
reaction was stirred for 2 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate and diluted with ethyl acetate. The organic
layer was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (compound 149, 6 mg, 0.0093
mmol, 64%). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 7.19 (s,
4H), 6.04-6.30 (m, 1H), 5.83-6.04 (m, 1H), 5.45-5.71 (m, 3H), 5.08
(d, J=10.8 Hz, 1H), 4.95 (d, J=9.5 Hz, 1H), 3.68 (br. s., 1H), 3.59
(s, 2H), 3.36-3.51 (m, 3H), 3.08-3.36 (m, 2H), 2.37-2.62 (m, 6H),
2.09-2.37 (m, 3H), 1.92-2.04 (m, 1H), 1.87 (br. s., 1H), 1.38-1.67
(m, 15H), 1.10-1.37 (m, 8H), 0.88-1.00 (m, 3H), 0.84 (d, J=6.8 Hz,
3H). MS (ES+)=647.5.
Compound 150 was prepared as shown in Scheme 15.
##STR00293##
Protocol for the Synthesis of Compound 150
[0715] Step 1: To a solution of a azide GGGG (8 mg, 0.013 mmol, 1.0
equiv.) in dichloromethane (1 mL, 0.02M) was added trimethyl
phosphine (1.0 molar solution, 0.026 mL, 0.026 mmol, 2.0 equiv) and
the reaction was heated at 50.degree. C. for 1 hour, or until the
reaction was determined to be complete by LCMS or TLC. Water (1.0
mL, 4.0 equiv.) was added and the reaction mixture was heated at
50.degree. C. for 3 hours, or until the reaction was determined to
be complete by LCMS or TLC. The solvent was removed to give the
crude amine ZZZZ (7.5 mg, 0.013 mmol, 98%).
[0716] Step 2: To a solution of amine ZZZZ (4.0 mg, 0.0068 mmol,
1.0 equiv.) in dichloromethane (0.5 mL, 0.01M) was added
triethylamine (0.005 mL, 0.029 mmol, 4.0 equiv.) at room
temperature. The reaction mixture was then cooled down to 0.degree.
C. and then cyclopentanecarbonyl chloride (0.0015 mL, 0.014 mmol,
2.0 equiv.) was added slowly. After warming up to room temperature,
the reaction was stirred for 3 hours, or until the reaction was
determined to be complete by LCMS or TLC. Upon completion of the
reaction, excess of solvent was removed and crude material was then
purified using silica gel chromatography (dichloromethane/methanol
as eluent) to afford the desired amide (AAAAA 2.42 mg, 0.0035 mmol,
52%).
[0717] Step 3: To a solution of amide AAAAA (21 mg, 0.026 mmol, 1.0
equiv.) in methanol (1 mL, 0.25M) at room temperature was added
p-methoxytoluenesulfonic acid (12.5 mg, 0.066 mmol, 2.5 equiv.).
The reaction was stirred for 3 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate and diluted with ethyl acetate. The organic
layer was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (compound 150, 12.9 mg, 0.019
mmol, 72%). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.91 (d,
J=6.65 Hz, 3H) 1.04 (d, J=6.65 Hz, 3H) 1.27 (s, 3H)) 1.20-1.62 (m,
18H) 1.65-1.78 (m, 5H) 1.79-1.87 (m, 2H) 190-2.01 (m, 2H) 2.37-2.67
(m, 5H) 2.72-2.84 (br.s., 4H) 2.87-2.98 (m, 1H) 3.04-3.14 (m, 1H)
3.25-3.36 (m, 1H) 3.66 (br. s., 4H) 3.71-3.80 (m, 1H) 5.02 (d,
J=9.41 Hz, 1H) 5.16 (d, J=10.67 Hz, 1H) 5.43 (t, J=5.34 Hz, 1H)
5.55-5.66 (m, 2H) 5.67 (dd, J=15.06, 9.29 Hz, 1H) 6.09 (d, J=11.17
Hz, 1H) 6.25 (dd, J=15.00, 10.85 Hz, 1H). MS (ES+)=686.6
[M+H].sup.+.
Compound 151 was prepared according to the method of Scheme 16.
##STR00294## ##STR00295##
Protocol for the Synthesis of Compound 151
[0718] Step 1: To a solution of amine WWW (95 mg, 0.38 mmol, 1.0
equiv.) in dichloromethane (4 mL, 0.1M) at 0.degree. C. was added
triethylamine (0.21 mL, 1.52 mmol, 4 equiv.) followed by
cyclopentanecarbonyl chloride (101 mg, 0.76 mmol, 2 equiv.). The
reaction was warmed to room temperature and was stirred for 10
hours, or until the reaction was determined to be complete by LCMS
or TLC. The reaction mixture was concentrated and the crude
material was then purified by silica gel column chromatography
(hexane/ethyl acetate) to give the desired amide (BBBBB, 49 mg,
0.14 mmol, 37%).
[0719] Step 2: To a solution of product BBBBB (49 mg, 0.14 mmol,
1.0 equiv.) in ethanol (4 mL, 0.03M) at 0.degree. C. was added
dropwise a premixed yellow solution of ammonium molybdate
tetrahydrate (33 mg, 0.028 mmol, 0.3 equiv.) in hydrogen peroxide
(0.15 mL, 1.4 mmol, 10 equiv. 33%). The reaction mixture was
allowed to warm up to room temperature and stirred for 4 hours, or
until the reaction was determined to be complete by LCMS or TLC.
The reaction mixture was diluted in ethyl acetate then sodium
thiosulfate was added at 0.degree. C. and the reaction was stirred
for 20 minutes. The organic layer was then washed with water,
brine, and dried over sodium sulfate. After evaporation of the
solvent, the residue was purified by silica gel column
chromatography (hexane/ethyl acetate) to give the desired sulfone
CCCCC (49 mg, 0.13 mmol, 92%).
[0720] Step 3: To a solution of sulfone CCCCC (13.6 mg, 0.36 mmol,
2.0 equiv.) in THF (15 mL, 0.01M) under nitrogen at -78.degree. C.
was added KHMDS (0.5 M in toluene, 0.14 mL, 0.072 mmol, 4.0 equiv.)
dropwise and the reaction was stirred for 20 minutes. Then aldehyde
E (10 mg, 0.018 mmol, 1.0 equiv.) in THF (0.5 M) was added
dropwise. The reaction was stirred at -78.degree. C. for 90 minutes
and then allowed to warm to -20.degree. C. for 1 hour. The reaction
was quenched with ammonium chloride and diluted with ethyl acetate.
The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product
(DDDDD, 4 mg, 0.0057 mmol, 31%).
[0721] Step 4: To a solution of product DDDDD (4 mg, 0.0057 mmol,
1.0 equiv.) in methanol (0.5 mL, 0.01M) was added potassium
carbonate (1.6 mg, 0.011 mmol, 2.0 equiv.) and the reaction was
stirred at room temperature. After 3 hours, or until the reaction
was determined to be complete by LCMS or TLC, the reaction was
quenched with ammonium chloride at 0.degree. C. The mixture was
then diluted with ethyl acetate. The organic layer was washed with
water, brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (hexane/ethyl acetate as eluent) to afford
the desired secondary alcohol (EEEEE, 4 mg, 0.0048 mmol, 85%).
[0722] Step 5: To a solution of alcohol EEEEE (4 mg, 0.006 mmol,
1.0 equiv.) in dichloromethane (0.7 mL, 0.006M) was added
triethylamine (0.0086 uL, 0.06 mmol, 10.0 equiv.), and DMAP (1.4
mg, 0.012 mmol, 2.0 equiv.) at 0.degree. C. Then a solution of
4-nitrophenyl carbonochloridate (4.86 mg, 0.024 mmol, 4.0 equiv.)
in dichloromethane (0.3 mL) was added slowly. The reaction was
warmed up to room temperature and stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate and diluted with
dichloromethane. The aqueous layer was extracted with
dichloromethane. The combined organic layers were washed with
brine, dried over sodium sulfate and concentrated in vacuo. The
crude protected carbonate (FFFFF) was used in the next step without
further purification.
[0723] Step 6: To a solution of carbonate FFFFF (1.0 equiv.) in THF
(1.0 mL, 0.06M) at room temperature was added N-methyl piperazine
(0.0067 uL, 10.0 equiv.). After stirring for one hour, or until the
reaction was determined to be complete by LCMS or TLC, the reaction
was quenched with water and diluted with ethyl acetate. The organic
layer was washed with 1N sodium hydroxide solution, and
concentrated. The resulting oil was purified by silica gel column
chromatography (dichloromethane/methanol as eluant) to afford the
desired product (GGGGG, 3 mg, 0.0038 mmol, 63%).
[0724] Step 7: To a solution of carbamate GGGGG (3.0 mg, 0.0038
mmol, 1.0 equiv.) in methanol (1 mL, 0.004M) at room temperature
was added p-methoxytoluenesulfonic acid (1.4 mg, 0.0076 mmol, 2.0
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(compound 151, 1.5 mg, 0.0022 mmol, 58%). .sup.1H NMR (400 MHz,
METHANOL-d4) .delta.: 0.88 (d, J=6.78 Hz, 3H) 1.02 (d, J=6.78 Hz,
3H) 1.21 (m, 3H) 1.27-1.44 (m, 6H) 1.53-1.72 (m, 6H) 1.74 (s, 3H)
1.76-1.84 (m, 2H) 2.35 (s, 3H) 2.41-2.63 (m, 10H) 3.05-3.17 (m, 2H)
3.54 (br.s., 1H) 3.79 (br. s., 1H) 4.94 (d, J=9.66 Hz, 1H) 5.04 (d,
J=10.54 Hz, 1H) 5.54-5.64 (m, 2H) 5.65-5.77(m, 1H) 6.02-6.13 (m,
1H) 6.22-6.34 (m, 1H) 7.78-7.86 (m, 1H), MS (ES+)=604.4
[M+H].sup.+. [0725] Compound 152 was prepared according to the
method of Scheme 17.
##STR00296##
[0725] Protocol for the Synthesis of Compound 152
[0726] Step 1: To a solution of 3-bromo-propan-1-ol HHHHH (2.0 g,
14.4 mmol, 1.0 equiv.) in dimethylformamide (40 mL, 0.3M) was added
imidazole (1.47 g, 21.6 mmol, 1.5 equiv.) followed by TBS-Cl (3.3
g, 21.6 mmol, 1.5 equiv.). The reaction was stirred at room
temperature for 6 hours, or until the reaction was determined to be
complete by LCMS or TLC. The reaction was then quenched with water.
The aqueous layer was then extracted with diethyl ether. The
combined organic layers were washed with brine and dried over
sodium sulfate. After filtration, the solvent was removed in vacuo
and the residue was purified by silica gel column chromatography
(hexane/ethyl acetate as eluant) to afford the desired protected
alcohol (IIIII, 2.91 g, 10.9 mmol, 76%).
[0727] Step 2: To a suspension of sodium hydride (55% suspension in
mineral oil, 0.32 g, 7.9 mmol, 1.0 equiv.) in DMF (15 mL, 0.5M) was
added 1-phenyl-1H-tetrazole-5-thiol (1.4 g, 7.9 mmol, 1.0 equiv.)
at 0.degree. C. The mixture was stirred for 1 hour. Then a solution
of (3-bromopropoxy)(tertbutyl)dimethylsilane IIIII (2.0 g, 7.9
mmol, 1.0 equiv.) was added to the reaction. The reaction was
heated to and maintained at 50.degree. C. for 10 hours, or until
the reaction was determined to be complete by LCMS or TLC. The
reaction was then quenched with water. The excess of DMF was
removed in vacuo. Then the residue was diluted in brine and
extracted with diethyl ether. The combined organic layers were
washed with brine, dried over MgSO4, filtered and the solvent
removed in vacuo. The residue was purified by silica gel column
chromatography (hexane/ethyl acetate) to afford the desired
terazole JJJJJ (2.5 g, 7.2 mmol, 91%).
[0728] Step 3: To a solution of the tetrazole JJJJJ (2.5 g, 7.2
mmol, 1.0 equiv.) in ethanol (45 mL, 0.1M) at 0.degree. C. was
added dropwise a premixed yellow solution of ammonium molybdate
tetrahydrate (0.89 g, 0.72 mmol, 0.1 equiv.) in hydrogen peroxide
(7.3 mL, 72 mmol, 10 equiv. 33%). The reaction mixture was allowed
to warm up to room temperature and stirred for 4 hours, or until
the reaction was determined to be complete by LCMS or TLC. The
reaction mixture was diluted in ethyl acetate, sodium thiosulfate
was added at 0.degree. C. and the reaction was stirred for 20
minutes. The organic layer was then washed with water, brine, and
dried over sodium sulfate. After evaporation of the solvent, the
residue was purified by silica gel column chromatography
(hexane/ethyl acetate) to give the desired sulfone (KKKKK, 1.7 g,
4.4 mmol, 62%).
[0729] Step 4: To a solution of the sulfone KKKKK (74 mg, 0.19
mmol, 2.5 equiv.) in THF (4 mL, 0.02M) under nitrogen at
-78.degree. C. was added KHMDS (0.5 M in toluene, 0.39 mL, 0.19
mmol, 2.5 equiv.) dropwise and the reaction was stirred for 20
minutes. Then aldehyde L (50 mg, 0.077 mmol, 1.0 equiv.) in THF (1
mL) was added dropwise. The reaction was stirred at -78.degree. C.
for 2 hours and then allowed to warm to -20.degree. C. for 1 hour.
The reaction was quenched with ammonium chloride and diluted with
ethyl acetate. The organic layer was washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product
(LLLLL, 30.6 mg, 0.038 mmol, 49%).
[0730] Step 5: To a solution of product LLLLL (30.6 mg, 0.038 mol,
1.0 equiv.) in methanol (3 mL, 0.01M) at room temperature was added
p-methoxytoluenesulfonic acid (7.2 mg, 0.038 mmol, 1.0 equiv.). The
reaction was stirred for 5 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate, diluted with ethyl acetate and washed with
water and brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (dichloromethane/methanol as eluent) to
afford the desired product (MMMMM, 10.7 mg, 0.019 mmol, 49%).
[0731] Step 6 and 7: To a stirred solution of product MMMMM (6.2
mg, 0.011 mmol, 1.0 equiv.) in 1,2-dichloromethane (1 mL, 0.01M) at
23.degree. C. was added DMAP (0.66 mg, 0.054 mmol, 0.5 equiv.) and
DIPEA (0.019 mL, 0.107 mmol, 10.0 equiv.). Then, 4-nitrophenyl
chloroformate (6.5 mg, 0.032 mmol, 3.0 equiv.) was added to the
mixture. After 16 hours, or until the reaction was determined to be
complete by LCMS or TLC, pyrrolidine (7.7 mg, 0.11 mmol, 10.0
equiv.) was added and the reaction was stirred for another 4 hours.
Dichloromethane was added to the reaction mixture. The organic
layer was then washed with water and brine. After drying over
sodium sulfate, filtration and evaporation, the crude material was
purified by silica gel column chromatography (ethyl
acetate/methanol) to give the desired product (compound 152, 2.67
mg, 0.0040 mmol, 37%). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.71-0.88 (m, 6H) 0.98-1.06 (m, 1H) 1.13-1.27 (m, 13H) 1.29-1.52
(m, 10H) 1.56 (br. s., 9H) 1.61-1.70 (m, 4H) 1.73 (br. s., 2H) 1.89
(br. s., 2H) 2.34-2.59 (m, 7H) 3.41 (d, J=5.27 Hz, 4H) 3.65-3.78
(m, 2H) 4.11 (t, J=6.78 Hz, 2H) 4.88-5.02 (m, 1H) 5.08 (d, J=10.54
Hz, 1H) 5.43-5.68 (m, 3H) 6.01 (d, J=10.29 Hz, 1H) 6.25 (dd,
J=15.06, 11.04 Hz, 1H), MS (ES+)=674.3 [M+14].sup.+. [0732]
Compound 153 was prepared according to the method of Scheme 18
##STR00297## ##STR00298##
[0732] Protocol for the Synthesis of Compound 153
[0733] Step 1: To a solution of
(S)-5-((3-((tert-butyldimethylsily0oxy)-2-methylpropyl)sulfonyl)-1-phenyl-
-1H-tetrazole (212 mg, 0.54 mmol, 2.5 equiv.) in THF (3 mL, 0.04M)
under nitrogen at -78.degree. C. was added KHMDS (0.5 M in toluene,
1.1 mL, 0.54 mmol, 2.5 equiv.) dropwise and the reaction was
stirred for 20 minutes. Then aldehyde Q (100 mg, 0.21 mmol, 1.0
equiv.) in THF (1 mL) was added dropwise. The reaction was stirred
at -78.degree. C. for 2 hours and then allowed to warm to
-20.degree. C. for 1 hour. The reaction was quenched with ammonium
chloride and diluted with ethyl acetate. The organic layer was
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel column chromatography (hexane/ethyl acetate as eluent) to
afford the desired product (NNNNN, 105 mg, 0.17 mmol, 77%).
[0734] Step 2: To a solution of product NNNNN (101 mg, 0.16 mmol,
1.0 equiv.) in methanol (4 mL, 0.04M) was added potassium carbonate
(55 mg, 0.40 mmol, 2.5 equiv.) and the reaction was stirred at room
temperature. After 3 hours, or until the reaction was determined to
be complete by LCMS or TLC, the reaction was quenched with ammonium
chloride at 0.degree. C. The mixture was then diluted with ethyl
acetate, washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (hexane/ethyl acetate as
eluent) to afford the desired secondary alcohol (OOOOO, 52 mg,
0.087 mmol, 55%).
[0735] Step 3: To a solution of alcohol OOOOO (20 mg, 0.034 mmol,
1.0 equiv.) in dichloromethane (4 mL, 0.01M) was added
triethylamine (0.033 mL, 0.24 mmol, 7.0 equiv.) and DMAP (1.2 mg,
0.01 mmol, 0.3 equiv.) at 0.degree. C. Then a solution of
4-nitrophenyl carbonochloridate (27.1 mg, 0.13 mmol, 4.0 equiv.) in
dichloromethane (1 mL) was added slowly. The reaction was warmed up
to room temperature and stirred for 3 hours or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
quenched with sodium bicarbonate and diluted with dichloromethane.
The aqueous layer was extracted with dichloromethane. The combined
organic layers were washed with brine, dried over sodium sulfate
and concentrated in vacuo. The crude protected carbonate (PPPPP)
was used in the next step without further purification.
[0736] Step 4: To a solution of carbonate PPPPP (1.0 equiv.) in THF
(4 mL, 0.01M) at room temperature was added N-methyl piperazine (34
mg, 0.34 mmol, 10.0 equiv.). After stirring for one hour, or until
the reaction was determined to be complete by LCMS or TLC, the
reaction was quenched with water and diluted with ethyl acetate.
The organic layer was washed with 1N sodium hydroxide solution
concentrated. The resulting oil was purified by silica gel column
chromatography (dichloromethane/methanol as eluant) to afford the
desired product (QQQQQ, 24 mg, 0.033 mmol, 99%).
[0737] Step 5: To a solution of product QQQQQ (24 mg, 0.033 mmol,
1.0 equiv.) in methanol (2 mL, 0.02M) at room temperature was added
p-methoxytoluenesulfonic acid (9.5 mg, 0.05 mmol, 1.5 equiv.). The
reaction was stirred for 5 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate and diluted with ethyl acetate. The organic
layer was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired product (RRRRR, 14.0 mg, 0.028 mmol,
85%).
[0738] Step 6: To a stirred solution of product RRRRR (4.0 mg,
0.008 mmol, 1.0 equiv.) in 1,2-dichloromethane (1.0 mL, 0.01M) at
23.degree. C. was added DMAP (0.2 mg, 0.0016 mmol, 0.2 equiv.) and
DIPEA (0.01 mL, 0.057 mmol, 7.0 equiv.). Then, 4-nitrophenyl
chloroformate (2.5 mg, 0.012 mmol, 1.5 equiv.) was added to the
mixture. After 12 hours, or until the reaction was determined to be
complete by LCMS or TLC, (R)-pyrrolidin-3-ol x (4.0 mg, 0.032 mmol,
4.0 equiv.) was added and stirred for another 4 hours.
Dichloromethane was added to the reaction mixture. The organic
layer was then washed with water and brine. After drying over
sodium sulfate, filtration and evaporation, the crude material was
purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to give the desired product
(compound 153, 3.2 mg, 0.0081 mmol, 65%). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.91 (d, J=6.78 Hz, 3H) 0.97-1.03 (m, 6H)
1.44 -1.55 (m, 2H) 1.67-1.85 (m, 5H) 1.86-2.01 (m, 2H) 2.37-2.62
(m, 11H) 3.02 (br. s., 1H) 3.20-3.32 (m, 1H) 3.38-3.63 (m, 9H)
3.68-3.75 (m, 2H) 3.79-4.10 (m, 2H) 4.40-4.48 (m, 1H) 4.86 (t,
J=10.10 Hz, 1H) 5.14 (dd, J=10.48, 5.58 Hz, 1H) 5.28-5.41 (m, 2H)
5.55 (dd, J=14.93, 9.91 Hz, 1H) 6.20 (t, J=11.23 Hz, 1H) 6.36 (br.
d, J=11.17 Hz, 1H). MS (ES+)=606.5 [MAH].sup.+. [0739] Compound 154
was prepared according to the method of Scheme 19.
##STR00299## ##STR00300##
[0739] Protocol for the Synthesis of Compound 154
[0740] Step 1: To a solution of
(R)--(S)-2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl
3-((tert-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate SSSSS
(100.0 mg, 0.20 mmol, 1.0 equiv.) in THF (4 mL, 0.04M) was added
slowly potassium bis(trimethylsilyl)amide (0.5 M in toluene, 1.2
mL, 0.59 mmol, 3 equiv.) at -78.degree. C. The reaction was stirred
for 15 minutes and (1H-benzo[d][1,2,3]triazol-1-yl)methanol (58.5
mg, 0.39 mmol, 2.0 equiv.) was added at -78.degree. C. The reaction
was stirred for 2 hours at -78.degree. C. and warmed to room
temperature over 1 hour. The reaction was quenched with ammonium
chloride, and extracted with ethyl acetate. The combined organic
layers were washed with brine, dried over sodium sulfate and
concentrated in vacuo. The crude material was then purified by
silica gel column chromatography (hexane/ethyl acetate as eluent)
to afford the desired terminal alkene (TTTTT, 40 mg, 0.13 mmol,
65%).
[0741] Step 2: To a solution of alkene TTTTT (40 mg, 0.13 mmol, 1.0
equiv.) in 1,2-dichloroethane (2.0 mL, 0.1M) was added
4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane (39.3 mg, 0.26
mmol, 2.0 equiv.). The reaction was purged with nitrogen and
Hoveyda-Grubbs II catalyst (8.0 mg, 0.013 mmol, 0.1 equiv.) was
added and the reaction was stirred at 50.degree. C. for 16 hours.
The mixture was filtered through Celite.RTM., the Celite.RTM. was
washed with dichloromethane and the filtrate was concentrated in
vacuo. The crude material was then purified by silica gel column
chromatography (hexane/ethyl acetate as eluent) to afford the
desired product (UUUUU, 31 mg, 0.063 mmol, 50%).
[0742] Step 3: To a solution of
(3S,4S,E)-1-iodo-2,4-dimethylhexa-1,5-dien-3-ol DD (240 mg, 0.95
mmol, 1.0 equiv.) in dichloromethane (7 mL, 0.2 M) was added
triphenylphosphine (400 mg, 1.5 mmol, 1.6 equiv.) and a solution of
NBS (288 mg, 1.6 mmol, 1.7 equiv.) in dichloromethane (1 mL, 0.1M)
using a syringe pump at 0.degree. C. The reaction was stirred at
0.degree. C. for 2 hours, or until the reaction was determined to
be complete by LCMS or TLC, before being quenched with a sodium
sulfite solution. The mixture was extracted with ethyl acetate,
washed with sodium bicarbonate, brine, and dried over sodium
sulfate. After evaporation of the solvent in vacuo, the crude
material was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired bromo
derivative (VVVVV, 270 mg, 0.86 mmol, 90%).
[0743] Step 4: To a solution of bromo derivative VVVVV (270 mg,
0.86 mmol, 1.0 equiv.) in DMF (5 mL, 0.2 M) was added sodium azide
(23 mg, 3.4 mmol, 4.0 equiv.). The reaction was heated to and
maintained at 50.degree. C. for 1 hour, or until the reaction was
determined to be complete by LCMS or TLC. Upon completion, the
reaction was cooled down to room temperature; the mixture was then
filtered through a silica gel plug (ethyl acetate). After
concentration, the azido derivative (WWWWW, 200 mg, 0.722 mmol,
84%) was used directly in the next step.
[0744] Step 5: To a solution of azide derivative WWWWW (22 mg,
0.079 mmol, 1.0 equiv.) in THF (2 mL, 0.04M) was added
trimethylphosphine (1.0 M, 0.16 mL, 0.6 mmol, 2.0 equiv.) at
-10.degree. C. The reaction was then warmed to room temperature and
stirred at room temperature for 30 minutes or until the reaction
was determined to be complete by LCMS or TLC. Then water (4.3 mL,
0.24 mmol, 3.0 equiv.) was added and the reaction was stirred at
room temperature for 5 hours, or until the reaction was determined
to be complete by LCMS or TLC. Upon completion, non-8-enoic acid
(25 mg, 0.16 mmol, 2.0 equiv.), HOBt (13 mg, 0.087 mmol, 1.1
equiv.), Hunig's base (0.047 mL, 0.32 mmol, 4.0 equiv.) and EDCI
(16.7 mg, 0.087 mmol, 1.1 equiv.) were added and the mixture was
stirred for 3 hours or until the reaction was determined to be
complete by LCMS or TLC. The solvent was then evaporated in vacuo,
ethyl acetate was added and the organic layer was extracted with
sodium bicarbonate and brine. After drying over sodium sulfate,
filtration and evaporation of solvent in vacuo, the crude material
was purified by silica gel chromatography (hexanes/ethyl acetate as
eluent) to afford the desired amide (XXXXX, 11 mg, 0.028 mmol,
36%).
[0745] Step 6: To a degassed solution of amide XXXXX (18 mg, 0.046
mmol, 1.0 equiv.) and benzoquinone (0.25 mg, 0.002 mmol, 0.05
equiv.) in toluene (4.6 mL, 0.01M) was added Hoveyda-Grubbs II
catalyst (2.9 mg, 0.0046 mmol, 0.1 equiv.). The mixture was stirred
in an oil bath at 65.degree. C. under a nitrogen atmosphere for 12
hours, or until the reaction was determined to be complete by LCMS
or TLC. The reaction mixture was cooled down to room temperature
and filtered through a Celite.RTM. and silica gel pad. The solvent
was then removed and the crude material was dissolved in dioxane (1
mL, 0.05M), and selenium dioxide (15.4 mg, 0.14 mmol, 3.0 equiv.)
was added. The mixture was heated to and maintained at 80.degree.
C. for 5 hours or until the reaction was determined to be complete
by LCMS or TLC. The mixture was cooled down to room temperature and
was diluted with ethyl acetate. The organic layer was washed with
sodium bicarbonate, brine and dried over sodium sulfate. The
solvent was removed in vacuo and the crude macrocycle was used in
the next step without further purification (YYYYY, 18 mg, 0.048
mmol).
[0746] Step 7: To a solution of macrocycle YYYYY (17 mg, 0.046 mmol
, 1.0 equiv.) in 1,2-dichloroethane (2 mL, 0.02 M) was added
nitrophenyl chloroformate (23.2 mg, 0.12 mmol, 2.5 equiv.),
triethylamine (0.045 mL, 0.322 mmol, 7.0 equiv.), and DMAP (5.6 mg,
0.046 mmol, 1.0 equiv.). The reaction was stirred at room
temperature for 12 hours or until the reaction was determined to be
complete by LCMS or TLC. N-methyl piperazine (14 mg, 0.14 mmol, 3.0
equiv.) was then added and the reaction was stirred at room
temperature for 2 hours, or until the reaction was determined to be
complete by LCMS or TLC. The mixture was then directly subjected to
purification by silica gel chromatography (dichloromethane/methanol
as eluent) to afford the desired carbamate (ZZZZZ, 15 mg, 0.029
mmol, 63%).
[0747] Step 8: To a solution of carbamate ZZZZZ (7 mg, 0.014 mmol,
1.0 equiv.) in THF (1 mL, 0.1M) at room temperature was added
(R)--(R,E)-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)but-3--
en-1 -yl 3-((tert-butyldimethylsilyl)oxy)pyrrolidine-1-carboxylate
UUUUU (8 mg, 0.018 mmol, 1.3 equiv.) , monosilver(I)
monosilver(III) monooxide (16 mg, 0.07 mmol, 5.0 equiv.), and
tetrakis(triphenylphosphine) palladium (0.8 mg, 0.007 mmol, 0.05
equiv.). The reaction mixture was heated to and maintained at
60.degree. C. for 30 minutes or until the reaction was determined
to be complete by LCMS or TLC. Upon completion, the reaction was
cooled down to room temperature, filtered through Celite.RTM.,
washed with dichloromethane and concentrated in vacuo. The crude
material was purified by silica gel chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(AAAAAA, 3.0 mg, 0.0044 mmol, 31%).
[0748] Step 8-2: To a solution of carbamate AAAAAA (3.0 mg, 0.0044
mmol, 1.0 equiv.) in methanol (1 mL, 0.004M) at room temperature
was added p-methoxytoluenesulfonic acid (4.1 mg, 0.022 mmol, 5.0
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(compound 154, 2.4 mg, 0.0042 mmol, 96%). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.97 (d, J=6.53 Hz, 3H) 1.07 (d, J=6.78 Hz,
3H) 1.31-1.70 (m, 7H) 1.71 (s, 3H) 1.80-2.06 (m, 3H) 2.21-2.32 (m,
2H) 2.32 (s, 3H) 2.39 (br. s., 4H) 2.56-2.64 (m, 1H) 3.42-3.59 (m,
6H) 3.91-4.02 (m, 2H) 4.15-4.26 (m, 1H) 4.45-4.50 (m, 1H) 5.14 (td,
J=10.04, 5.14 Hz, 1H) 5.26-5.40 (m, 2H) 5.58 (dd, J=15.06, 10.04
Hz, 1H) 5.65 (dd, J=15.12, 7.47 Hz, 1H) 6.06 (d, J=10.41 Hz, 1H)
6.27 (dd, J=14.62, 11.23 Hz, 1H). MS (ES+)=575.4 [M+H].sup.+.
[0749] Compound 155 was prepared according to the method of Scheme
20.
##STR00301##
[0749] Protocol for the Synthesis of Compound 155
[0750] Step 1: To a solution of macrocycle
(8R,11S,12S,E)-8-hydroxy-12-((E)-1-iodoprop-1-en-2-yl)-11-methyloxacyclod-
odec-9-en-2-one GG (0.20 g, 0.53 mmol, 1.0 equiv.) in
dichloromethane (5.3 mL, 0.1M) was added PPh3 (0.28 g, 1.0 mmol,
2.0 equiv.) and CBr4 (0.35 g, 1.0 mmol, 2.0 equiv.) at 0.degree. C.
The reaction was stirred at room temperature for 3 hours, or until
the reaction was determined to be complete by LCMS or TLC . The
reaction mixture was then quenched with water and aqueous layer
extracted with dichloromethane. The combined organic layers were
then washed with brine, dried over MgSO4 and filtered. The solvent
was removed in vacuo and the residue was then diluted in DMF (5.3
mL, 0.1M). Sodium azide (0.14 g, 2.1 mmol, 4.0 equiv.) was added
and the reaction was warmed to 70.degree. C. for 12 hours, or until
the reaction was determined to be complete by LCMS or TLC. Upon
completion, the solvent was removed and the crude material was
purified by silica gel column chromatography (hexanes/ethyl acetate
as eluent) to afford the desired azide (BBBBBB, 0.17 g, 0.20 mmol,
39%).
[0751] Step 2: To a solution of azide BBBBBB (0.16 g, 0.20 mmol,
1.0 equiv.) in THF (0.1M) was added trimethyl phosphine (0.035 mL,
0.40 mmol, 2.0 equiv.) and the reaction was stirred at 50.degree.
C. for 1 hours, or until the reaction was determined to be complete
by LCMS or TLC. Water (0.014 mL, 0.8 mmol, 4.0 equiv.) was added
and the reaction mixture was heated at 50.degree. C. for 3 hours,
or until the reaction was determined to be complete by LCMS or TLC.
The solvent was removed and the crude material was purified by
silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired amine (CCCCCC, 0.06 g, 0.16 mmol,
79%).
[0752] Step 3: To a solution of amine CCCCCC (0.08 g, 0.21 mmol,
1.0 equiv.) in dichloromethane (2.0 mL, 0.1M) at 0.degree. C. was
added triethylamine (0.12 mL, 0.85 mmol, 4.0 equiv.), DMAP (26.1
mg, 0.21 mmol, 1.0 equiv.) followed by 4-methylpiperazine-1
-carbonyl chloride (0.07 g, 0.43 mmol, 2.0 equiv.). The reaction
was warmed to room temperature and was stirred for 7 hours, or
until the reaction was determined to be complete by LCMS or TLC.
The reaction was quenched with sodium bicarbonate. The organic
layer was washed with water and brine. After drying over sodium
sulfate and filtration, the solvent was removed in vacuo. The
residue was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired urea
(DDDDDD, 0.069 g, 0.14 mmol, 65%).
[0753] Step 4: To a solution of DDDDDD (3.0 mg, 0.006 mmol, 1.0
equiv.) in THF (0.5 mL, 0.1M) at room temperature was added
(R,E)-2-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)but-3-en-1--
yl pyrrolidine-1-carboxylate SSSSS (3.7 mg, 0.012 mmol, 2.0
equiv.), monosilver(I) monosilver(III) monooxide (6.9 mg, 0.03
mmol, 5.0 equiv.), triphenylarsine (2.2 mg, 0.007 mmol, 1.2
equiv.), and tetrakis(triphenylphosphine) palladium (0.82 mg,
0.009, 0.15 equiv.). The reaction mixture was heated at 60.degree.
C. for 30 minutes, or until the reaction was determined to be
complete by LCMS or TLC. Upon completion, the reaction was cooled
down to room temperature, the mixture was then filtered through
Celite.RTM., the Celite.RTM. was washed with dichloromethane and
concentrated in vacuo. The crude material was purified by silica
gel chromatography (dichloromethane/methanol as eluent) to afford
the desired product (Compound 155, 2.7 mg, 0.0048 mmol, 81%).
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.78-0.97 (m, 6H) 1.06
(d, J=6.78 Hz, 3H) 1.16-1.39 (m, 4H) 1.43-1.61 (m, 2H) 1.76-1.87
(m, 2H) 2.22-2.47 (m, 7H) 2.55-2.64 (m, 1H) 3.30-3.41 (m, 8H)
3.90-4.00 (m, 2H) 4.15-4.28 (m, 4H) 5.02 (d, J=10.67 Hz, 1H)
5.14-5.30 (m, 3H) 5.35-5.45 (m, 2H) 5.67 (dd, J=15.06, 7.53 Hz, 1H)
6.10 (d, J=11.42 Hz, 1H) 6.23-6.30 (m, 1H) 6.45 (d, J=0.88 Hz, 1H),
MS (ES+)=559.5 [M+H].sup.+. [0754] Compound 156 was prepared
according to the method of Scheme 21.
##STR00302## ##STR00303##
[0754] Protocol for the Synthesis of Compound 156
[0755] Step 1: To a solution of
(S)-5-((3-((tert-butyldimethylsilyl)oxy)-2-methylpropyl)sulfonyl)-1-pheny-
l-1H-tetrazole (0.066 g, 0.17 mmol, 2.0 equiv.) in THF (2.0 mL,
0.04M) under nitrogen at -78.degree. C. was added KHMDS (0.33 mL,
0.17 mmol, 2.0 equiv.) dropwise and the reaction was stirred for 20
minutes. Then aldehyde D (0.040 g, 0.08 mmol, 1.0 equiv.) in THF
(0.2 mL) was added dropwise. The reaction was stirred at
-78.degree. C. for 2 hours and then allowed to warm to -20.degree.
C. over 1 hour. The reaction was quenched with ammonium chloride
and diluted with ethyl acetate. The organic layer was washed with
water, brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (hexane/ethyl acetate as eluent) to afford
the desired product (EEEEEE, 0.036 g, 0.06 mmol, 68%).
[0756] Step 2: To a solution of EEEEEE (0.037 g, 0.06 mmol, 1.0
equiv.) in methanol (2.0 mL, 0.03M) at room temperature was added
pyridinium p-toluenesulfonate (0.015 g, 0.06 mmol, 1.0 equiv.). The
reaction was stirred for 6 hours, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with sodium bicarbonate and diluted with ethyl acetate. The organic
layer was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (dichloromethane/methanol as
eluent) to afford the desired primary alcohol (FFFFFF, 0.02 g, 0.04
mmol, 68%).
[0757] Step 3: To a solution of primary alcohol FFFFFF (5.0 mg,
0.009 mmol, 1.0 equiv.) and acid
(2R,35)-2-methyl-3-((triethylsilyl)oxy)pentanoic acid (3.5 mg,
0.014 mmol, 1.5 equiv.) in dichloromethane (0.3 mL, 0.03M) was
added diisopropylethyamine (0.003 mL, 0.02 mmol, 2.0 equiv.), DMAP
(1.1 mg, 0.009 mmol, 1.0 equiv.) and COMU (6.0 mg, 0.014 mmol, 1.5
equiv.) at 0.degree. C. The reaction mixture was warmed up to room
temperature and stirred for 16 hours or until the reaction was
determined to be complete by LCMS or TLC. Upon completion, the
reaction was diluted with dichloromethane, washed with water, brine
and dried over sodium sulfate. After filtration, and evaporation,
the crude material was purified by silica gel column chromatography
(hexanes/ethyl acetate as eluent) to afford the desired ester
GGGGGG (5.0 mg, 0.006 mmol, 70%).
[0758] Step 4: To a solution of ester GGGGGG (4.0 mg, 0.005 mmol,
1.0 equiv.) in methanol (0.1 mL, 0.005M) at room temperature was
added p-methoxytoluenesulfonic acid (1.0 mg, 0.005 mmol, 1.0
equiv.). The reaction was stirred for 3 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(dichloromethane/methanol as eluent) to afford the desired product
(compound 156, 1.2 mg, 0.002 mmol, 40%). .sup.1H NMR (400 MHz,
METHANOL, d4) .delta.: 6.60 (dd, J=15.1, 11.0 Hz, 1H), 6.17 (d,
J=11.0 Hz, 1H), 5.86 (dt, J=15.2, 6.3 Hz, 1H), 5.66-5.78 (m, 1H),
4.96-5.19 (m, 2H), 4.67 (d, J=6.5 Hz, 1H), 3.78-4.00 (m, 1H), 3.73
(ddd, J=8.3, 5.8, 4.5 Hz, 1H), 3.15 (s, 1H), 2.44-2.67 (m, 3H),
2.07-2.10 (m, 2H), 1.75-1.86 (m, 2H), 1.57-1.67 (m, 1H), 1.37-1.55
(m, 3H), 1.15-1.24 (m, 4H), 0.82-1.07 (m, 5H), MS (ES+)=561.3
[M+Na].sup.+. [0759] Compound 157 was prepared by the method of
Scheme 22.
##STR00304##
[0760] Step 1: Intermediate EEEEEE (40.6 mg, 0.062 mmol, 1.0 equiv)
was dissolved in EtOH (2 mL) and PPTS (1.562 mg, 6.217 .mu.mol, 0.1
equiv) was added. The reaction mixture was stirred for 1 hr at rt.
Then, the solvent was removed. The residue was purified by silica
gel chromatography (25-80% EtOAc/hexanes) to give desired product
(Compound 157, 2.7 mg, 6.36 .mu.mol, 10%). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: ppm 0.72-0.89 (m, 3H) 0.97 (d, J=6.78 Hz,
3H) 1.11-1.35 (m, 7H) 1.42-1.56 (m, 2H) 1.56-1.71 (m, 4H) 2.02 (s,
3H) 2.34-2.58 (m, 3H) 3.32-3.54 (m, 3H) 3.56-3.83 (m, 1H) 5.01 (d,
J=9.03 Hz, 1H) 5.09 (d, J=10.54 Hz, 1H) 5.50-5.65 (m, 3H) 6.03 (d,
J=10.79 Hz, 1H) 6.25 (ddd, J=15.06, 10.79, 1.00 Hz, 1H). MS(ES+):
425.30 [M+H].sup.+. [0761] Compounds 158-160 were prepared by the
method of Scheme 23.
##STR00305##
[0761] General Protocol for the Synthesis of Compounds 158-160
[0762] Step 1: To a solution of the corresponding sulfone (2.5
equiv.) in THF (0.02M) under nitrogen at -78.degree. C. was added
KHMDS (2.5 equiv.) dropwise and stirred for 20 minutes. Then
aldehyde L (1.0 equiv.) in THF (0.5 M) was added dropwise. The
reaction was stirred at -78.degree. C. for 2 hours and then allowed
to warm to -20.degree. C. over 1 hour. The reaction was quenched
with ammonium chloride and diluted with ethyl acetate. The organic
layer was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (hexane/ethyl acetate as
eluent) to afford the desired product (HHHHHH)
[0763] Step 2: To a solution of carbamate HHHHHH (1.0 equiv.) in
methanol (0.1M) at room temperature was added pyridinium
p-toluenesulfonate (5.0 equiv.). The reaction was stirred for 6
hours, or until the reaction was determined to be complete by LCMS
or TLC. The reaction was quenched with sodium bicarbonate and
diluted with ethyl acetate. The organic layer was washed with
water, brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The resulting oil was purified by silica gel
column chromatography (hexanes/ethyl acetate as eluent) to afford
the desired carbamate (compounds 158-160). The two diastereoisomers
could be isolated after preparative HPLC. Column: Waters Xbridge
C18 5 .mu.m OBD 19.times.150 mm. Mobile phase A: 0.1% NH.sub.4OH in
water (pH 10), Mobile phase B: 0.1% NH.sub.4OH in 100%
acetonitrile. Mobile phase conditions: isocratic 45% B in 10 min 30
mL/min.
Exemplified Protocol for the Synthesis of Compound 90 and
separation of the two epimers, Compound 159 and Compound 160
[0764] Step 1: To a solution
3-(1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridine
(0.15 g, 0.45 mmol, 2.1 equiv.) in THF (4.8 mL, 0.04M) under
nitrogen at -78.degree. C. was added KHMDS (0.46 mL, 0.46 mmol, 2.2
equiv.) dropwise and the reaction was stirred for 20 minutes. Then
aldehyde L (0.13 g, 0.21 mmol, 1.0 equiv.) in THF (0.2 mL) was
added dropwise. The reaction was stirred at -78.degree. C. for 2
hours and then allowed to warm to -20.degree. C. for 1 hour. The
reaction was quenched with ammonium chloride and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product
(HHHHHH, 0.08 g, 0.11 mmol, 53%).
[0765] Step 2: To a solution of carbamate HHHHHH (0.08 g, 0.11
mmol, 1.0 equiv.) in methanol (1.0 mL, 0.1M) at room temperature
was added pyridinium p-toluenesulfonate (0.1 g, 0.55 mmol, 5.0
equiv.). The reaction was stirred for 6 hours, or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with sodium bicarbonate and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexanes/ethyl acetate as eluent) to afford the desired carbamate
(compound 90, 0.015 g, 0.02 mmol, 22%) as a mixture of epimers at
C16. .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.70-0.93 (m, 4H)
1.00 (d, J=6.78 Hz, 1H) 1.12-1.21 (m, 5H) 1.24-1.50 (m, 14H)
1.55-1.76 (m, 11H) 1.90 (br. s., 2H) 2.14 (s, 1H) 2.36-2.57 (m, 7H)
3.22-3.43 (m, 5H) 3.43-3.59 (m, 1H) 3.68 (br. s., 1H) 4.94 (d,
J=9.54 Hz, 1H) 5.08 (d, J=10.79 Hz, 1H) 5.44-5.72 (m, 2H) 5.82 (dd,
J=15.43, 6.90 Hz, 1H) 6.03 (d, J=10.54 Hz, 1H) 6.09-6.25 (m, 1H)
7.16-7.21 (m, 3H) 7.44 (d, J=7.28 Hz, 1H) 8.41 (br. s., 2H), MS
(ES+)=638.8 [M+H].sup.+.
[0766] The mixture was then subjected to preparative HPLC
separation using the following parameters: Column: Waters Xbridge
C18 5 .mu.m OBD 19.times.150 mm. Mobile phase A: 0.1% NH.sub.4OH in
water (pH 10), Mobile phase B: 0.1% NH.sub.4OH in 100%
acetonitrile. Mobile phase conditions: isocratic 45% B in 10 min 30
mL/min. fraction 1, rt=5.9 min, fraction 2, rt=6.9 min.
[0767] Compound 159 (fraction 1, WiDr GI.sub.50=13.3 nM, Panc05.04
GI.sub.50=15.0 nM) .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.82-1.00 (m, 3H) 1.24-1.31 (m, 4H) 1.34-1.60 (m, 12H) 1.65-1.88
(m, 8H) 1.96-2.12 (m, 1H) 2.45-2.67 (m, 7H) 3.50 (br. s., 4H) 3.59
(t, J=7.03 Hz, 1H) 3.68-3.88 (m, 1H) 4.95-5.10 (m, 1H) 5.17 (d,
J=10.54 Hz, 1H) 5.54-5.79 (m, 2H) 5.90 (dd, J=15.06, 7.03 Hz, 1H)
6.12 (d, =10.79 Hz, 1H) 6.27 (ddd, J=15.06, 10.79, 1.25 Hz, 1H)
7.26 (d, J=4.77 Hz, 1H) 7.54 (dt, J=4.51 Hz, 1H) 8.41-8.58 (m,
2H).
[0768] Compound 160 (fraction 2, WiDr GI.sub.50=29.5 nM, Panc05.04
GI.sub.50=15.8 nM) .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.87-0.97 (m, 3H) 1.23-1.30 (m, 4H) 1.32-1.60 (m, 12H) 1.64-1.78
(m, 7H) 1.78-1.93 (m, 2H) 1.99 (br. s., 1H) 2.42-2.68 (m, 6H)
3.37-3.64 (m, 5H) 3.76 (d, J=6.53 Hz, 1H) 5.03 (d, J=9.54 Hz, 1H)
5.17 (d, J=10.54 Hz, 1H) 5.54-5.78 (m, 2H) 5.91 (dd, J=14.93, 6.90
Hz, 1H) 6.12 (d, J=11.54 Hz, 1H) 6.18-6.40 (m, 1H) 7.27 (s, 1H)
7.41-7.66 (m, 1H) 8.40-8.60 (m, 2H). [0769] Carbamate (Scheme 24)
and Heterocycle (Scheme 25) Sidechain Julia Fragments Synthesis
##STR00306##
[0769] General Protocol for the Synthesis of Carbamate Julia
Fragment
[0770] Step 1: To a solution of (S)-3-bromo-2-methylpropanol SSS
(10.0 g, 65.3 mmol, 1.0 equiv.) in dichloromethane (300 mL, 0.1M)
at 0.degree. C. was added imidazole (6.7 g, 98.0 mmol, 1.5 equiv.)
followed by TBSC1 (11.8 g, 78.4 mmol, 1.2 equiv.). The reaction was
allowed to warm to room temperature and stirred at room temperature
overnight. Once determined to be complete by TLC or LCMS, the
reaction was filtered. The filtrate was washed with water,
saturated sodium bicarbonate, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The crude material was
purified by silica gel column chromatography (hexane/ethyl acetate)
to give the desired protected alcohol (IIIIII, 13.5 g, 50.5 mmol,
77%).
[0771] Step 2: To a solution of NaH (2.4 g, 60.6 mmol, 1.2 equiv.)
in DMF (200 mL, 0.2M) at 0.degree. C. was added
1-phenyl-1H-tetrazole-5-thiol (9.9 g, 55.6 mmol, 1.1 equiv.) and
the reaction was stirred for 1 hour. Next, a solution of bromide
HIM (13.5 g, 50.5 mmol, 1.0 equiv.) was added at 0.degree. C. and
the reaction was gradually heated to 80.degree. C. for ten hours.
Once determined to be complete by TLC or LCMS, the reaction was
cooled to 0.degree. C. and quenched with water. The reaction was
concentrated and the resulting oil was purified by silica gel
column chromatography (hexane/ethyl acetate as eluent) to afford
the desired product (JJJJJJ, 15.6 g, 42.8 mmol, 85%).
[0772] Step 3: To a solution of tetrazole JJJJJJ (2.4 g, 6.5 mmol,
1.0 equiv.) in ethanol (60 mL, 0.1M) at 0.degree. C. was added
ammonium molybdate tetrahydrate (0.8 g, 0.65 mmol, 0.1 equiv.) and
hydrogen peroxide (6.6 mL, 64.7 mmol, 10.0 equiv., 30% solution in
water). The reaction was allowed to warm to room temperature and
stirred at room temperature for four hours or until the reaction
was determined to be complete by LCMS or TLC. The reaction was
quenched with water and diluted with ethyl acetate. The organic
layer was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The resulting oil was purified
by silica gel column chromatography (hexane/ethyl acetate as
eluent) to afford the desired sulfone (KKKKKK, 2.2 g, 5.4 mmol,
84%).
[0773] Step 4: To a solution of sulfone KKKKKK (1.0 g, 2.5 mmol,
1.0 equiv.) in methanol (25.0 mL, 0.1M) at room temperature was
added p-toluenesulfonic acid (0.1 g, 0.5 mmol, 0.2 equiv.). The
reaction was stirred for 1 hour, or until the reaction was
determined to be complete by LCMS or TLC. The reaction was quenched
with aqueous sodium bicarbonate solution, and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The crude
product (LLLLLL, 0.7 g, 2.5 mmol, 100%) was advanced without
further purification.
[0774] Step 5: To a solution of alcohol LLLLLL (1.0 equiv.) in
dichloromethane (0.1M) at -10.degree. C. was added DMAP (1.1
equiv.), DIEA (1.1 equiv.) and 4-nitrophenylchloroformate (1.1
equiv.). The reaction was allowed to warm to room temperature and
stirred at room temperature overnight or until the reaction was
determined to be complete by LCMS or TLC. Next, the reaction was
cooled to 0.degree. C. and the corresponding amine was added. The
reaction was allowed to warm to room temperature and stirred at
room temperature for five hours. The reaction was diluted with
ethyl acetate and washed with water, brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The resulting oil was
purified by silica gel column chromatography (hexane/ethyl acetate
as eluent) to afford the desired sulfone (SSSSS).
Exemplified Protocol for the Synthesis of
(S)-2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl
pyrrolidine-1-carboxylate
[0775] Steps 1-4 as above.
[0776] Step 5: To a solution of alcohol LLLLLL (0.25 g, 0.89 mmol,
1.0 equiv.) in dichloromethane (1.5 mL, 0.5M) at -10.degree. C. was
added DMAP (0.16 g, 1.3 mmol, 1.5 equiv.), DIEA (1.2 mL, 7.09 mmol,
8.0 equiv.) and 4-nitrophenyl chloroformate (0.7 g, 3.5 mmol, 4.0
equiv.). The reaction was allowed to warm to room temperature and
stirred at room temperature overnight or until the reaction was
determined to be complete by LCMS or TLC. Next, the reaction was
cooled to 0.degree. C. and pyrrolidine (0.37 mL, 4.4 mmol, 5
equiv.) was added. The reaction was allowed to warm to room
temperature and stirred at room temperature for five hours. The
reaction was diluted with ethyl acetate and washed with water,
brine, dried over magnesium sulfate, filtered, and concentrated in
vacuo. The resulting oil was purified by silica gel column
chromatography (hexane/ethyl acetate as eluent) to afford the
desired product
(S)-2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propyl
pyrrolidine-1-carboxylate (0.32 g, 0.84 mmol, 95%). .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 7.67-7.77 (m, 2H), 7.59-7.67 (m,
3H), 4.18-4.29 (m, 1H), 4.12 (dd, J=14.7, 4.3 Hz, 1H), 3.98 (dd,
J=11.0, 7.0 Hz, 1H), 3.58 (dd, J=14.7, 8.2 Hz, 1H), 3.34-3.46 (m,
4H), 2.61-2.74 (m, 1H), 1.83-1.98 (m, 4H), 1.23 (d, J=6.9 Hz, 3H).
[0777] Protocol for the Synthesis of
2-(3-methyl-1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)butan-2-yl)pyridine
##STR00307##
[0778] Step 1: NNNNNN (704 mg, 4.657 mmol, 1 equiv) was dissolved
in THF (22.100 mL) at 0.degree. C. and sodium tert-butoxide (470
mg, 4.89 mmol, 1.05 equiv) was added. This reaction solution turned
bright yellow and was stirred for 30 min at this tempurature. Then,
2-iodopropane (0.931 mL, 9.314 mmol, 2 equiv) was added and the
reaction solution was stirred at rt for 3 hrs. The reaction mixture
was quenched with sat. aq. ammonium chloride and the THF was
evaporated by rotavap. The remaining aqueous was extracted with
EtOAc twice and the combined organics were washed with brine, dried
over sodium sulfate, filtered, and concentrated to give the desired
crude product (SPE-30, 477.5 mg, 2.471 mmol, 53.1%).
[0779] Step 2: SPE-30 (477.5 mg, 2.471 mmol, 1 equiv) was dissolved
in THF (25.300 mL) at 0.degree. C. and lithium aluminium hydride
(2.97 mL, 2.965 mmol, 1.2 equiv) was added dropwise. The reaction
mixture was warmed to rt over 30 min then stirred at rt. The
reaction mixture was carefully quenched with water, sodium
hydroxide, and water, then stirred for 30 min. The ppt was filtered
off and the solvent evaporated. The residue was extracted with
ether and the combined organics were washed with water and brine
then dried over sodium sulfate, filtered, and concentrated to give
the crude desired product (SPE-31, 238 mg, 1.441 mmol, 58%).
[0780] Step 3: SPE-31 (238 mg, 1.44 mmol, 1 equiv) was dissolved in
DCM (8805 .mu.L) at 0.degree. C. and triethylamine (221 .mu.l,
1.584 mmol, 1.1 equiv) was added. Mesyl chloride (118 .mu.L, 1.512
mmol, 1.05 equiv) was added dropwise and the reaction mixture
stirred at this temperature for 30 min. The reaction was quenched
with sat. aq. sodium bicarbonate and the aqueous re-extracted with
DCM. The combined organics were washed with brine, dried over
sodium sulfate, filtered, and concentrated to give crude desired
product (SPE-32, 202 mg, 0.830 mmol, 57.6%).
[0781] Step 4: SPE-32 (202 mg, 0.83 mmol, 1 equiv) was dissolved in
DMF (8035 .mu.L) at rt and cesium carbonate (379 mg, 1.162 mmol,
1.4 equiv) was added followed by 1-phenyl-1H-tetrazole-5-thiol (178
mg, 0.996 mmol, 1.2 equiv). The mixture was stirred at 50.degree.
C. for 72 hrs. Brine was added and the aqueous layer was extracted
3.times. with ether. The combined organics were washed with water
and brine then dried over sodium sulfate, filtered, and
concentrated. Purification by column chromatography (0-100%
EtOAc/hexanes) was completed to give the desired product (SPE-33,
130.3 mg, 0.400 mmol, 48.2%).
[0782] Step 5: SPE-33 (130.3 mg, 0.40 mmol, 1 equiv) was suspended
in EtOH (2922 .mu.L) at -10.degree. C. and ammonium molybdate
tetrahydrate (24.74 mg, 0.02 mmol, 0.05 equiv) was added followed
by hydrogen peroxide (204 .mu.L, 2.002 mmol, 5 equiv). The reaction
mixture was stirred at this temperature for 3 hrs. Then, 3 mL THF
was added and the reaction mixture was stirred at rt for 36 hrs.
The mixture was quenched with water and aq. sodium metabisulfite.
The reaction was diluted with EtOAc, the layers separated, then the
organics washed with aq. sodium thiosulfate and water. The organics
were dried over sodium sulfate, filtered, and concentrated.
Purification by column chromatography (0-100% EtOAc/hexanes) was
completed to give the desired product (SPE-9, 92 mg, 0.257 mmol,
64.3% yield). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: ppm
0.78-0.88 (m, 3H) 0.93-1.02 (m, 3H) 2.02-2.17 (m, 1H) 3.29-3.44 (m,
1H) 3.94-4.06 (m, 1H) 4.59-4.72 (m, 1H) 7.10-7.19 (m, 2H) 7.60 (s,
6H) 8.38-8.47 (m, 1H). MS(ES+): 358.30 [M+H].sup.+. [0783] Protocol
for the Synthesis of Compound 161
##STR00308##
[0784] Step 1: To a solution of SPE-9 (93 mg, 0.261 mmol, 1.8
equiv) in 1:4 DMF (247 .mu.L)/ THF (998 pt) at -78.degree. C. was
added slowly 0.6 M NaHMDS (375 .mu.l, 0.225 mmol, 1.55M) so as to
maintain the reaction temperature below -70.degree. C. This was
stirred for 30 min at this temperature. To this cooled yellow
solution was added dropwise slowly a solution of aldehyde D (70 mg,
0.145 mmol, 1 equiv) in THF (198 .mu.L). The reaction temperature
was maintained below -65.degree. C. The aldehyde vessel was rinsed
with THF and added dropwise to the cooled reaction mixture. This
was then stirred between -70 to -60.degree. C. for 1 hr (set
cryocoil to -65.degree. C.). Then, the cryocoil was set to
-50.degree. C. and the reaction mixture was let stir at that
temperature o/n. The reaction mixture was warmed to -40.degree. C.
and solid ammonium chloride (33.9 mg, 0.634 mmol, 4.37 equiv) was
added. The reaction was further warmed to 0.degree. C. and water
was added followed by toluene. The aqueous layer was separated and
the organic layer was then washed with brine. The organics were
dried over sodium sulfate, filtered, and concentrated. Purification
by column chromatography (0-40% MTBE/hexanes with long hold at 40%
MTBE/hexanes to elute product) was completed to give desired
product (SPE-10, 29 mg, 0.047 mmol, 32.6%).
[0785] Step 2: SPE-10 (29 mg, 0.047 mmol, 1 equiv) was dissolved in
THF (240 .mu.L) and TBAF (94 pt, 0.094 mmol, 2 equiv) was added.
The solution was stirred at rt o/n. The solution was diluted with
water and extracted with EtOAc. The organics were then washed with
brine and the organics were dried over sodium sulfate, filtered,
and concentrated. Purification by column chromatography (0-100%
EtOAc/hexanes) was completed to give desired product (Compound 161,
14.4 mg, 0.028 mmol, 61%). .sup.1H NMR (400 MHz, METHANOL-d4)
.delta.: ppm 0.76 (dd, J=6.59, 1.32 Hz, 3H) 0.78-0.87 (m, 1H) 0.91
(d, J=6.65 Hz, 2H) 0.99 (d, J=6.78 Hz, 3H) 1.20 (s, 3H) 1.33-1.43
(m, 2H) 1.55-1.65 (m, 2H) 1.70-1.79 (m, 3H) 2.00-2.04 (m, 1H)
2.05-2.19 (m, 4H) 2.53 (br dd, J=15.75, 3.33 Hz, 3H) 3.11-3.22 (m,
1H) 3.73-3.85 (m, 1H) 5.03-5.09 (m, 2H) 5.51-5.63 (m, 1H) 5.66-5.76
(m, 1H) 5.99 (dd, J=15.00, 9.60 Hz, 1H) 6.13 (br d, J=10.79 Hz, 1H)
6.32-6.43 (m, 1H) 7.23-7.38 (m, 2H) 7.72-7.83 (m, 1H) 8.41-8.52 (m,
1H). MS(ES+): 500.58 [M+H].sup.+. [0786] General Protocol for the
Synthesis of
(S)-2-(1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridine
##STR00309##
[0787] Step 1: To a solution of 2-(pyridin-2-yl)acetic acid
hydrochloride salt MMMMMM (50.0 g, 288.0 mmol, 1.0 equiv.) in
methanol (500 mL, 0.5M) at 0.degree. C. was added thionyl chloride
(31.5 mL, 432.0 mmol, 1.5 equiv.) dropwise. The reaction was
stirred at 0.degree. C. for 60 minutes or until the reaction was
determined to be complete by LCMS or TLC. The reaction was
carefully quenched with sodium carbonate and the aqueous layer
extracted with ethyl acetate. The combined organic layers were
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting product (NNNNNN, 41.5 g,
275.0 mmol, 95%) was used in the next step without further
purification.
[0788] Step 2: To a solution of ester NNNNNN (41.5 g, 275.0 mmol,
1.0 equiv.) in THF (1500 mL, 0.2M) at 0.degree. C. was added sodium
2-methylpropan-2-olate (28.6 g, 288.3 mmol, 1.05 equiv.) and the
reaction mixture was stirred for 30 minutes at 0.degree. C. before
addition of iodomethane (34.3 mL, 549.1 mmol, 2.0 equiv.). The
reaction was stirred at room temperature for 1 hour or until the
reaction was determined to be complete by LCMS or TLC. The reaction
was quenched with ammonium chloride and the excess of solvent was
removed in vacuo. The crude material was then extracted with ethyl
acetate. The combined organic layers were washed with brine, and
dried over magnesium sulfate. After filtration and evaporation of
the solvent, the mixture was concentrated in vacuo. The resulting
methyl ester (OOOOOO, 41.3 g, 250 mmol, 91%) was advanced without
purification.
[0789] Step 3: To a solution of methyl ester OOOOOO (43.0 g, 260.3
mmol, 1.0 equiv.) in THF (1500 mL, 0.1M) at 0.degree. C. was added
lithium aluminum hydride (312 mL, 312.4 mmol, 1.2 equiv., solution
in THF) dropwise. The reaction was allowed to warm gradually to
0.degree. C. for 30 minutes and then to room temperature for 1 hour
or until the reaction was determined to be complete by LCMS or TLC.
The reaction was carefully quenched with water, sodium hydroxyde
and water. After stirring the mixture for 30 minutes, the white
precipitate was filtered off and the solvent was removed in vacuo.
The reaction was then extracted with diethyl ether and the combined
organic fractions were washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting alcohol (PPPPPP, 30.0 g, 219.0 mmol, 84%) was advanced
without purification.
[0790] Step 4: To a solution of alcohol PPPPPP (30.0 g, 219.0 mmol,
1.0 equiv.) in dichloromethane (700 mL, 0.3M) at 0.degree. C. was
added triethylamine (61.5 mL, 437.4 mmol, 2.0 equiv), and DMAP (2.7
g, 21.9 mmol, 0.1 equiv.). Acetic anhydride (24.8 mL, 262.4 mmol,
1.2 equiv.) was added and the reaction mixture was stirred for 30
minutes or until the reaction was determined to be complete by LCMS
or TLC. The reaction was quenched with ammonium chloride, and
organic layer washed with brine, dried over magnesium sulfate and
filtered. The resulting solution was then evaporated and the crude
acetate (QQQQQQ, 37.0 g, 206.0 mmol, 94%) was used in the following
step without further purification.
[0791] Step 5: A solution of acetate QQQQQQ (39.4 g, 219.8 mmol,
1.0 equiv.) was dissolved in diethyl ether (100 mL) and then 118 g
of silica gel was added. The excess of ether was removed in vacuo
and the crude solid was then diluted in pH 7 aqueous buffer (1970
mL, 0.1M). (sodium hydroxyde/sodium phosphate monobasic/water) Then
porcine pancreatic lipase type II (3.3 g, (15 mg/mmol)) was added
and the reaction was stirred at 37.degree. C. for four hours or
until determined to be complete by TLC or LCMS. (After four hours,
conversion reached 40% according to ELSD and the enantiomeric
excess was determined by chiral SFC, and showed an enantiomeric
ratio of 13:1 S:R). The silica gel was filtered off and the aqueous
layer was extracted with ethyl acetate three times. The combined
organic layers were washed with brine, dried over magnesium sulfate
and concentrated. The product was purified by silica gel column
chromatography (hexanes:ethyl acetate as eluant) to afford the
desired alcohol (RRRRRR, 12.5 g, 91 mmol, 41%).
[0792] Step 6: To a solution of alcohol RRRRRR (12.5 g, 91.0 mmol,
1.00 equiv.) in dichloromethane (570 mL, 0.16M) at room temperature
was added triethylamine (13.9 mL, 100.1 mmol, 1.1 equiv). The
reaction was cooled down to 0.degree. C. and then methanesulfonyl
chloride (7.44 mL, 95.5 mmol, 1.05 equiv) was added. The reaction
was stirred at 0.degree. C. for 30 minutes or until determined to
be complete by TLC or LCMS. The reaction was quenched with sodium
bicarbonate and the layers were separated. The aqueous layer was
then extracted with dichloromethane. The combined organic layers
were washed with brine, dried over magnesium sulfate, and
concentrated in vacuo. The resulting sulfonate SSSSSS (19.2 g, 89
mmol, 98%) was advanced without additional purification.
[0793] Step 7: To a solution of sulfonate SSSSSS (19.2 g, 89 mmol,
1.0 equiv.) in DMF (120 mL, 0.1M) at room temperature was added
cesium carbonate (40.7 g, 125.0 mmol, 1.4 equiv.) and
1-phenyl-1H-tetrazole-5-thiol (19.1 g, 107.1 mmol, 1.2 equiv.). The
resulting mixture was stirred at 50.degree. C. for 48 hours, or
until determined to be complete by TLC or LCMS. After cooling the
mixture to room temperature, brine was added and the aqueous layer
was extracted three times with diethyl ether. The combined organic
layers were washed with water, brine, and dried over magnesium
sulfate. After filtration, the solvent was removed in vacuo and the
residue was purified using silica gel column chromatography
(hexanes/ethyl acetate) to give the desired product (TTTTTT, 28.9
g, 88 mmol, 99%).
[0794] Step 8: To a solution of sulfide TTTTTT (31.5 g, 105.9 mmol,
1.0 equiv.) in EtOH (700 mL, 0.1M) at -10.degree. C. was added
ammonium molybdate tetrahydrate (6.5 g, 5.3 mmol, 0.05 equiv.) and
hydrogen peroxide (108 mL, 1060 mmol, 5.0 equiv., 33% aqueous
solution). The reaction was stirred at -10.degree. C. for four
hours or until determined to be complete by TLC or LCMS. The
reaction was quenched with water and sodium metabisulfite solution.
The crude product was collected by filtration and was purified by
silica gel column chromatography (hexanes:ethyl acetate as eluant)
to afford the desired product (UUUUUU, 23.2 g, 70.4 mmol, 66%).
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 1.50 (d, J=7.03 Hz,
3H) 1.66 (br. s., 1H) 3.75 (quind, 1H) 3.94 (dd, J=14.81, 5.02Hz,
1H) 4.55 (dd, J=14.68, 7.91 Hz, 1H) 7.14-7.22 (m, 2H) 7.29 (s, 1H)
7.57-7.70 (m, 6H) 8.44-8.49 (m, 1H).
[0795] Racemic
2-(1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridine
could be prepared using a similar synthetic strategy as described
in scheme 23 by skipping steps 5 and 6 (Lipase resolution).
[0796] Other heterocyclic Julia fragments, including
(3-(1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridine,
4-(1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridine,
4-(2-methyl-3-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyrimidine-
, and
3-(1(1-((1-phenyl-1H-tetrazol-5-yl)sulfonyl)propan-2-yl)pyridazine,
were prepared in a similar manner (steps 5 and 6 (lipase
resolution) were skipped to produce a racemic mixture at C16)
starting with the corresponding heterocycle. [0797] Preparation of
racemic Compounds 36 and 37
##STR00310##
[0798] Step SN-1: To a stirred solution of sn-2 (2.55 equiv.) in
THF (30 mL) at -78.degree. C. under N.sub.2 was added KHMDS (2.55
equiv., 0.5M solution in toluene) slowly. The reaction was stirred
at -78.degree. C. for 30 minutes. Next, aldehyde sn-1 (1g, 1.89
mmol, 1.0 equiv.) in THF (10 mL, final conc. 0.047M) was added
slowly at -78.degree. C. and the reaction was stirred for 3.5 hours
at the same temperature, or until the reaction was determined to be
complete by LCMS or TLC. The reaction was allowed to warm to room
temperature. The reaction mixture was diluted with water and ethyl
acetate. The aqueous layer was extracted with additional ethyl
acetate and the combined organic layers were washed with brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(heptane/ethyl acetate as eluent) to afford the desired product
sn-3 (1.08g, 90%). LCMS data (ES+) M+Na 654.4.
[0799] Step SN-2: A stirred solution of the protected macrolide
sn-3 (530 mg, 0.837 mmol, 1.0 equiv.) in acetic acid/water (4:1)
(0.042M) was heated at 80.degree. C. for 8 hours under N.sub.2. The
reaction mixture was evaporated and the resulting residue was
dissolved with water and ethyl acetate. The aqueous solution was
adjusted to pH=9 by the addition of saturated aqueous NaHCO.sub.3
solution. The resulting aqueous layer was extracted with additional
ethyl acetate. The combined organic layers were washed with brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
(heptane/ethyl acetate as eluent) to afford the desired product
sn-4 (127 mg, 35%). LCMS data (ES+) M+430.2.
[0800] Step SN-3: To a stirred solution of the triol sn-4 (127 mg,
0.296 mmol, 1.0 equiv.) in dichloromethane (0.05M) at 0.degree. C.
under N.sub.2, triethylamine (2 equiv.), acetic anhydride (1
equiv.) and 4-dimethylaminopyridine (0.2 equiv.) were added. The
resulting mixture was stirred at 0.degree. C. for 1 hour or until
the reaction was determined to be complete by LCMS or TLC. The
reaction was quenched with saturated aqueous NaHCO.sub.3 solution
and ethyl acetate was added. The aqueous layer was extracted with
ethyl acetate. The combined organic layers were washed with water
and brine, dried over magnesium sulfate, filtered, and concentrated
in vacuo. The residue was purified by silica gel column
chromatography (heptane/ethyl acetate as eluent) to afford the
mixture of Compounds 36 and 37 (112 mg, 80%). [0801] .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.83-0.96 (m, 3H) 1.17-1.82 (m,
13H) 2.08 (s, 3H) 2.43-2.70 (m, 3H) 3.40-3.83 (m, 3H) 5.07 (d,
J=8.8 Hz, 1H) 5.14 (d, J=10.8 Hz, 1H) 5.53-5.73 (m, 2H) 5.92-6.05
(m, 1H) 6.07-6.18 (m, 1H) 6.25-6.38 (m, 1H) 7.06-7.21 (m, 2H)
7.56-7.69 (m, 1H) 8.48-8.60 (m, 1H). [0802] LCMS data (ES+) M+Na
494.1.
[0803] The intermediate of macrolide aldehyde sn-1 was prepared as
previously reported (R. M. Kanada and D. Ito et. al., Angew. Chem.
Int. Ed. 2007, 46, 4350-4355), and sn-2 was prepared in an
analogous manner as described in Scheme 23. [0804] Protocol for
Synthesis of Compound 162
##STR00311##
[0805] Step 1: To a solution of E7107 (30 mg, 0.042 mmol, 1.0
equiv) in DCE (1 mL) was added DMAP (1.020 mg, 8.34 .mu.m, 0.2
equiv), Hunig's base (0.037 mL, 0.209 mmol, 5.0 equiv) and acetic
anhydride (4.72 .mu.L, 0.05 mmol, 1.2 equiv). After 2 hrs, the
reaction mixture was evaporated. Purification by silica gel
chromatography (0-10% MeOH/DCM) was completed to give desired
product (Compound 162, 24 mg, 0.032 mmol, 76%). .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: ppm 0.01 (d, J=4.52 Hz, 6H) 0.76-0.88
(m, 14H) 0.99 (d, J=6.78 Hz, 3H) 1.14 (s, 3H) 1.18-1.25 (m, 3H)
1.31-1.59 (m, 8H) 1.63 (d, J=0.75 Hz, 4H) 2.03 (s, 4H) 2.27-2.58
(m, 4H) 2.71-2.86 (m, 3H) 3.10-3.24 (m, 2H) 3.71-3.82 (m, 1H) 3.89
(d, J=6.78 Hz, 2H) 4.89 (d, J=10.67 Hz, 1H) 5.01 (d, J=9.29 Hz, 1H)
5.50-5.65 (m, 3H) 6.05 (s, 1H) 6.12-6.28 (m, 1H). MS(ES+): 761.73
[M+H].sup.+.
TABLE-US-00011 TABLE 8 Compounds 147-162 LCMS data Structure,
Compound #, and Chemical Name .sup.1H NMR data (ES+) ##STR00312##
.sup.1H NMR (400 MHz, METHANOL- d4) 0.78-0.98 (m, 6 H) 0.99-1.06
(m, 3 H) 1.19-1.28 (m, 4 H) 1.28- 1.43 (m, 2 H) 1.50-1.62 (m, 14 H)
1.63-1.75 (m, 6 H) 1.85-1.99 (m, 2 H) 2.41-2.68 (m, 7 H) 2.73-2.89
(m, 1 H) 3.39-3.62 (m, 4 H) 3.75 (br. s., 2 H) 4.07-4.26 (m, 2 H)
5.01 (d, J = 9.54 Hz, 1 H) 5.13 (d, J = 10.67 Hz, 1 H) 5.55-5.74
(m, 3 H) 6.01- 6.07 (m, 1 H) 6.10-6.20 (m, 1 H) 683.5 ##STR00313##
.sup.1H NMR (400 MHz, METHANOL- d4) .delta.: 0.91 (d, J = 6.65 Hz,
3 H) 1.09 (d, J = 6.78 Hz, 3 H) 1.11-1.19 (m, 1 H) 1.23 (s, 3 H)
1.30-1.46 (m, 3 H) 1.50-1.69 (m, 2 H) 1.77 (s, 3 H) 2.40 (s, 3 H)
2.46-2.71 (m, 7 H) 3.41- 3.70 (m, 4 H) 3.75 (s, 3 H) 3.78-3.84 (m,
1 H) 3.92-4.09 (m, 2 H) 4.96 (d, J = 9.54 Hz, 1 H) 5.07 (d, J =
10.67 Hz, 1 H) 5.53-5.81 (m, 3 H) 6.11 (d, J = 10.54 Hz, 1 H) 6.38
(dd, J = 15.00, 10.85 Hz, 1 H) 566.5 ##STR00314## .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 7.19 (s, 4H), 6.04-6.30 (m, 1H),
5.83-6.04 (m, 1H), 5.45-5.71 (m, 3H), 5.08 (d, J = 10.8 Hz, 1H),
4.95 (d, J = 9.5 Hz, 1H), 3.68 (br. s., 1H), 3.59 (s, 2H),
3.36-3.51 (m, 3H), 3.08-3.36 (m, 2H), 2.37-2.62 (m, 6H), 2.09-2.37
(m, 3H), 1.92-2.04 (m, 1H), 1.87 (br. s., 1H), 1.38-1.67 (m, 15H),
1.10- 1.37 (m, 8H), 0.88-1.00 (m, 3H), 0.84 (d, J = 6.8 Hz, 3H)
647.5 ##STR00315## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.91 (d, J = 6.65 Hz, 3 H) 1.04 (d, J = 6.65 Hz, 3 H) 1.27 (s, 3
H)) 1.20-1.62 (m, 18 H) 1.65-1.78 (m, 5 H) 1.79-1.87 (m, 2 H)
190-2.01 (m, 2 H) 2.37- 2.67 (m, 5 H) 2.72-2.84 (br. s., 4 H)
2.87-2.98 (m, 1 H) 3.04-3.14 (m, 1 H) 3.25-3.36 (m, 1 H) 3.66 (br.
s., 4 H) 3.71-3.80 (m, 1 H) 5.02 (d, J = 9.41 Hz, 1 H) 5.16 (d, J =
10.67 Hz, 1 H) 5.43 (t, J = 5.34 Hz, 1 H) 5.55- 5.66 (m, 2 H) 5.67
(dd, J = 15.06, 9.29 Hz, 1 H) 6.09 (d, J = 11.17 Hz, 1 H) 6.25 (dd,
J = 15.00, 10.85 Hz, 1 H) 686.6 ##STR00316## .sup.1H NMR (400 MHz,
METHANOL- d4) 0.88 (d, J = 6.78 Hz, 3 H) 1.02 (d, J = 6.78 Hz, 3 H)
1.21 (m, 3 H) 1.27- 1.44 (m, 6 H) 1.53-1.72 (m, 6 H) 1.74 (s, 3 H)
1.76-1.84 (m, 2 H) 2.35 (s, 3 H) 2.41-2.63 (m, 10 H) 3.05- 3.17 (m,
2 H) 3.54 (br. s., 1 H) 3.79 (br. s., 1 H) 4.94 (d, J = 9.66 Hz, 1
H) 5.04 (d, J = 10.54 Hz, 1 H) 5.54-5.64 (m, 2 H) 5.65-5.77 (m, 1
H) 6.02- 6.13 (m, 1 H) 6.22-6.34 (m, 1 H) 7.78-7.86 (m, 1 H) 604.4
##STR00317## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.71-0.88
(m, 6 H) 0.98-1.06 (m, 1 H) 1.13- 1.27 (m, 13 H) 1.29-1.52 (m, 10
H) 1.56 (br. s., 9 H) 1.61-1.70 (m, 4 H) 1.73 (br. s., 2 H) 1.89
(br. s., 2 H) 2.34-2.59 (m, 7 H) 3.41 (d, J = 5.27 Hz, 4 H)
3.65-3.78 (m, 2 H) 4.11 (t, J = 6.78 Hz, 2 H) 4.88-5.02 (m, 1 H)
5.08 (d, J = 10.54 Hz, 1 H) 5.43-5.68 (m, 3 H) 6.01 (d, J = 10.29
Hz, 1 H) 6.25 (dd, J = 15.06, 11.04 Hz, 1 H) 674.3 ##STR00318##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.91 (d, J = 6.78 Hz,
3 H) 0.97-1.03 (m, 6 H) 1.44-1.55 (m, 2 H) 1.67-1.85 (m, 5 H)
1.86-2.01 (m, 2 H) 2.37-2.62 (m, 11 H) 3.02 (br. s., 1 H) 3.20-
3.32 (m, 1 H) 3.38-3.63 (m, 9 H) 3.68-3.75 (m, 2 H) 3.79-4.10 (m, 2
H) 4.40-4.48 (m, 1 H) 4.86 (t, J = 10.10 Hz, 1 H) 5.14 (dd, J =
10.48, 5.58 Hz, 1 H) 5.28-5.41 (m, 2 H) 5.55 (dd, J = 14.93, 9.91
Hz, 1 H) 6.20 (t, J = 11.23 Hz, 1 H) 6.36 (br. d, J = 11.17 Hz, 1
H) 606.5 ##STR00319## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.97 (d, J = 6.53 Hz, 3 H) 1.07 (d, J = 6.78 Hz, 3 H) 1.31-1.70 (m,
7 H) 1.71 (s, 3 H) 1.80-2.06 (m, 3 H) 2.21-2.32 (m, 2 H) 2.32 (s, 3
H) 2.39 (br. s., 4 H) 2.56-2.64 (m, 1 H) 3.42-3.59 (m, 6 H)
3.91-4.02 (m, 2 H) 4.15- 4.26 (m, 1 H) 4.45-4.50 (m, 1 H) 5.14 (td,
J = 10.04, 5.14 Hz, 1 H) 5.26- 5.40 (m, 2 H) 5.58 (dd, J = 15.06,
10.04 Hz, 1 H) 5.65 (dd, J = 15.12, 7.47 Hz, 1 H) 6.06 (d, J =
10.41 Hz, 1 H) 6.27 (dd, J = 14.62, 11.23 Hz, 1 H) 575.4
##STR00320## .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.78-0.97
(m, 6 H) 1.06 (d, J = 6.78 Hz, 3 H) 1.16-1.39 (m, 4 H) 1.43-1.61
(m, 2 H) 1.76-1.87 (m, 2 H) 2.22-2.47 (m, 7 H) 2.55-2.64 (m, 1 H)
3.30- 3.41 (m, 8 H) 3.90-4.00 (m, 2 H) 4.15-4.28 (m, 4 H) 5.02 (d,
J = 10.67 Hz, 1 H) 5.14-5.30 (m, 3 H) 5.35- 5.45 (m, 2 H) 5.67 (dd,
J = 15.06, 7.53 Hz, 1 H) 6.10 (d, J = 11.42 Hz, 1 H) 6.23-6.30 (m,
1 H) 6.45 (d, J = 0.88 Hz, 1 H) 559.5 ##STR00321## .sup.1H NMR (400
MHz, METHANOL- d4) .delta.: 0.79 (d, J = 6.8 Hz, 2 H) 0.84 (t, J =
7.5 HZ, 1H) 0.97 (d, J = 6.78 Hz, 1 H) 1.05-1.09 (m, 2 H) 1.14-
1.32 (m, 5 H) 1.36 (dt, J = 7.84, 3.98 Hz, 1 H) 1.44-1.59 (m, 1 H)
1.65 (d, J = 1.00 Hz, 2 H) 1.96 (s, 2 H) 2.16- 2.39 (m, 3 H) 3.03
(dt, J = 3.51, 2.01 Hz, 2 H) 3.38 (dt, J = 3.26, 1.63 Hz, 1 H)
3.46-3.63 (m, 1 H) 3.69 (br. s., 1 H) 3.78-4.04 (m, 1 H) 4.46 (br.
s., 1 H) 4.67 (s, 1 H) 4.95 (d, J = 10.04 Hz, 2 H) 5.46-5.65 (m, 2
H) 5.99 (d, J = 12.05 Hz, 1 H) 6.25 (dd, J = 15.94, 10.92 Hz, 1 H)
(M + Na) 561.3 ##STR00322## .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta.: ppm 0.72- 0.89 (m, 3 H) 0.97 (d, J = 6.78 Hz, 3 H)
1.11-1.35 (m, 7 H) 1.42-1.56 (m, 2 H) 1.56-1.71 (m, 4 H) 2.02 (s, 3
H) 2.34-2.58 (m, 3 H) 3.32-3.54 (m, 3 H) 3.56-3.83 (m, 1 H) 5.01
(d, J = 9.03 Hz, 1 H) 5.09 (d, J = 10.54 Hz, 1 H) 5.50-5.65 (m, 3
H) 6.03 (d, J = 10.79 Hz, 1 H) 6.25 (ddd, J = 15.06, 10.79, 1.00
Hz, 1 H) 425.30 ##STR00323## .sup.1H NMR (400 MHz,
DICHLOROMETHANE-d2) .delta.: 8.48 (d, J = 3.6 Hz, 2H), 7.14 (d, J =
4.6 Hz, 2H), 6.31 (dddd, J = 15.2, 10.8, 4.5, 1.3 Hz, 1H), 6.10
(dd, J = 10.8, 1.0 Hz, 1H), 5.87 (dd, J = 15.0, 7.5 Hz, 1H),
5.63-5.74 (m, J = 9.7 Hz, 1H), 5.55 (ddd, J = 15.4, 10.2, 1.8 Hz,
1H), 5.13 (d, J = 10.7 Hz, 1H), 4.97 (d, J = 9.7 Hz, 1H), 3.64-3.78
(m, 1H), 3.53 (quin, J = 7.0 Hz, 1H), 3.41 (br. s., 4H), 3.31 (br.
d, J = 7.2 Hz, 1H), 2.55 (d, J = 3.5 Hz, 3H), 2.46 (br. s., 4H),
1.74 (d, J = 1.3 Hz, 4H), 1.70- 1.81 (m, 4H), 1.59-1.70 (m, 3H),
1.39 (d, J = 13.3 Hz, 1H), 1.34-1.58 (m, 11H), 1.25-1.34 (m, 3H),
1.20 (s, 3H), 0.88 (dd, J = 8.2, 6.9 Hz, 3H) 638.5 ##STR00324##
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.82-1.00 (m, 3 H)
1.24-1.31 (m, 4 H) 1.34- 1.60 (m, 12 H) 1.65-1.88 (m, 8 H)
1.96-2.12 (m, 1 H) 2.45-2.67 (m, 7 H) 3.50 (br. s., 4 H) 3.59 (t, J
= 7.03 Hz, 1 H) 3.68-3.88 (m, 1 H) 4.95- 5.10 (m, 1 H) 5.17 (d, J =
10.54 Hz, 1 H), 5.54-5.79 (m, 2 H) 5.90 (dd, J = 15.06, 7.03 Hz, 1
H) 6.12 (d, = 10.79 Hz, 1 H) 6.27 (ddd, J = 15.06, 10.79, 1.25 Hz,
1 H) 7.26 (d, J = 4.77 Hz, 1 H) 7.54 (dt, J = 4.51 Hz, 1 H)
8.41-8.58 (m, 2 H) 638.5 ##STR00325## .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.87-0.97 (m, 3 H) 1.23-1.30 (m, 4 H) 1.32-
1.60 (m, 12 H) 1.64-1.78 (m, 7 H) 1.78-1.93 (m, 2 H) 1.99 (br. s.,
1 H) 2.42-2.68 (m, 6 H) 3.37-3.64 (m, 5 H) 3.76 (d, J = 6.53 Hz, 1
H) 5.03 (d, J = 9.54 Hz, 1 H) 5.17 (d, J = 10.54 Hz, 1 H) 5.54-5.78
(m, 2 H) 5.91 (dd, J = 14.93, 6.90 Hz, 1 H) 6.12 (d, J = 11.54 Hz,
1 H) 6.18-6.40 (m, 1 H) 7.27 (s, 1 H) 7.41-7.66 (m, 1 H) 8.40-8.60
(m, 2 H) 638.4 ##STR00326## .sup.1H NMR (400 MHz, METHANOL- d4)
.delta.: ppm 0.76 (dd, J = 6.59, 1.32 Hz, 3 H) 0.78-0.87 (m, 1 H)
0.91 (d, J = 6.65 Hz, 2 H) 0.99 (d, J = 6.78 Hz, 3 H) 1.20 (s, 3 H)
1.33-1.43 (m, 2 H) 1.55-1.65 (m, 2 H) 1.70-1.79 (m, 3 H) 2.00-2.04
(m, 1 H) 2.05- 2.19 (m, 4 H) 2.53 (br dd, J = 15.75, 3.33 Hz, 3 H)
3.11-3.22 (m, 1 H) 3.73-3.85 (m, 1 H) 5.03-5.09 (m, 2 H) 5.51-5.63
(m, 1 H) 5.66-5.76 (m, 1 H) 5.99 (dd, J = 15.00, 9.60 Hz, 1 H) 6.13
(br d, J = 10.79 Hz, 1 H) 6.32-6.43 (m, 1 H) 7.23-7.38 (m, 2 H)
7.72-7.83 (m, 1 H) 8.41-8.52 (m, 1 H) 500.58 ##STR00327## .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta.: ppm 0.01 (d, J = 4.52 Hz, 6 H)
0.76-0.88 (m, 14 H) 0.99 (d, J = 6.78 Hz, 3 H) 1.14 (s, 3 H)
1.18-1.25 (m, 3 H) 1.31-1.59 (m, 8 H) 1.63 (d, J = 0.75 Hz, 4 H)
2.03 (s, 4 H) 2.27-2.58 (m, 4 H) 2.71- 2.86 (m, 3 H) 3.10-3.24 (m,
2 H) 3.71-3.82 (m, 1 H) 3.89 (d, J = 6.78 Hz, 2 H) 4.89 (d, J =
10.67 Hz, 1 H) 5.01 (d, J = 9.29 Hz, 1 H) 5.50-5.65 (m, 3 H) 6.05
(s, 1 H) 6.12-6.28 (m, 1 H) 761.73
Synthesis of Compounds 163-174
[0806] Synthesis of Compound 163
##STR00328##
[0807] Step MM-1: To a mixture of macrolide diene x (15 mg, 0.041
mmol, 1.0 equiv.) and commercially available allylic alcohol m-12
(3.0 equiv.) in dichloromethane (0.014M) was added Hoveyda-Grubbs
II catalyst (0.1 equiv.). The reaction mixture was stirred at
reflux under a nitrogen atmosphere for 1 hour, or until the
reaction was determined to be completed by TLC. The reaction
mixture was cooled to room temperature and concentrated in vacuo.
The crude product was purified by silica gel column chromatography
(heptane:ethyl acetate as eluant) to afford the title Compound 163
(12.6 mg, 59%). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.90(d, J=6.4 Hz, 3H) 1.21(s, 3H) 1.23-1.47(m, 5H) 1.48-1.74(m, 4H)
1.77(s, 3H) 1.79-1.94(m, 2H) 2.10(s, 3H) 2.47-2.74(m, 5H) 3.54(d,
J=10.4 Hz, 1H) 3.75(br. s., 1H) 5.09(d, J=8.8 Hz, 1H) 5.18(d,
J=10.8 Hz, 1H) 5.58-5.72(m, 2H) 5.86(d, J=15.2 Hz, 1H) 6.13(d,
J=10.8 Hz, 1H) 6.48(dd, J=15.2, 10.8 Hz, 1H) 7.10-7.22(m, 3H)
7.23-7.31(m, 2H). LCMS data (ES+) M+Na 537.3.
[0808] The intermediate diene x was prepared as previously reported
(R. M. Kanada and D. Ito et. al., Angew. Chem. Int. Ed. 2007, 46,
4350-4355). [0809] Synthesis of Compound 164
##STR00329##
[0810] The title Compound 164 (6.8 mg, 61%) was prepared from
commercially available allylic alcohol m-12 (6.0 equiv.) and
macrolide diene x (8.4 mg, 0.23 mmol, 1.0 equiv.) in an analogous
manner as described for step MM-1. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.80-0.95(m, 3H) 1.15-1.46(m, 5H)
1.60-1.78(m, 9H) 1.90 (s, 1H) 2.10 (s, 3H) 2.45-2.67 (m, 3H) 3.51
(d, J=10.8 Hz, 1H) 3.76 (br. s., 1H) 5.09 (d, J=8.8 Hz, 1H) 5.17
(d, J=10.4 Hz, 1H) 5.57-5.71 (m, 2H) 6.06 (d, J=14.8 Hz, 1H) 6.14
(d, J=10.0 Hz, 1H) 6.50 (ddd, J=15.2, 10.8, 8.4 Hz, 1H) 7.21-7.30
(m, 1H) 7.31-7.39 (m, 2H) 7.40-7.49 (m, 2H). LCMS data (ES+) M+Na
509.3. [0811] Synthesis of Compound 165
##STR00330##
[0812] The title Compound 165 (5.3 mg, 46%) was prepared from
commercially available thiophene sn-5 (29.9 mg, 0.194 mmol, 8.3
equiv.) and macrolide diene x (8.6 mg, 0.0235 mmol, 1.0 equiv.) in
an analogous manner as described for MM-1. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 0.82-0.98 (m, 3H) 1.15-1.44 (m, 8H)
1.47-1.88 (m, 5H) 1.99-2.26 (m, 5H) 2.44-2.74 (m, 3H) 3.44-3.61 (m,
1H) 3.75 (br. s., 1H) 5.02-5.26 (m, 2H) 5.53-5.75 (m, 2H) 6.00-6.22
(m, 2H) 6.47-6.63 (m, 1H) 6.89-7.03 (m, 2H) 7.17-7.35 (m, 1H). LCMS
data (ES+) M+Na 515.1. [0813] Synthesis of Compound 166
##STR00331##
[0814] The title Compound 166 was prepared in an analogous manner
to previously described reaction of Julia fragments with aldehydes.
The intermediate of macrolide aldehyde sn-1 was prepared as
previously reported (R. M. Kanada and D. Ito et. al., Angew. Chem.
Int. Ed. 2007, 46, 4350-4355). [0815] The sulfone intermediate m-11
was prepared in the following manner.
##STR00332##
[0816] The sulfone intermediate m-11 (48 mg, 22% in 2 steps) was
prepared from commercially available alcohol m-10 in an analogous
manner as described in Scheme 23. LCMS data (ES+) M+Na 319.06.
[0817] Synthesis of Compound 167
##STR00333##
[0818] Coupling product m-12 (62 mg, 99%) was prepared from
aldehyde sn-1 (50 mg, 0.095 mmol, 1.0 equiv.) and sulfone m-9 (2.5
equiv.) in an analogous manner as described for step SN-1. LCMS
data (ES+) M+Na 684.31. Triol m-13 (21 mg, 49%) was prepared in an
analogous manner as described for step SN-2.
[0819] The title Compound 167 (2.8 mg, 12%) was prepared from triol
m-13 in an analogous manner as described for step SN-3. .sup.1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.80-0.95(m, 3H) 1.21 (s, 3H)
1.24-1.73 (m, 8H) 1.74 (s, 3H) 2.09 (s, 3H) 2.42-2.67 (m, 3H)
3.48-3.62 (m, 2H) 3.74 (br. s., 1H) 3.93 (s, 3H) 5.08 (d, J=8.40
Hz, 1H) 5.16 (d, J=10.40 Hz, 1H) 5.57-5.70 (m, 2H) 5.99-6.07 (m,
1H) 6.11 (d, J=10.80 Hz, 1H) 6.25-6.34 (m, 1H) 6.54 (d, J=7.20 Hz,
1H) 6.70 (d, J=7.20 Hz, 1H) 7.48 (t, J=8.00 Hz, 1H). LCMS data
(ES+) M+Na 524.2. [0820] The sulfone intermediate m-9 was prepared
in the following manner.
##STR00334##
[0821] Compound m-8 (426 mg, 63% in 2 steps) was prepared in an
analogous manner as described for step mm-3 and step mm-4. LCMS
data (ES+) M+Na 189.95.
[0822] Compound m-9 (490 mg, 65% in 2 steps) was prepared in an
analogous manner as described in Scheme 23. LCMS data (ES+) M+Na
381.98. [0823] Synthesis of Compound 168
##STR00335##
[0824] Coupling product m-15 (35 mg, 52%) was prepared from
aldehyde sn-1 (50 mg, 0.095 mmol, 1.0 equiv.) and sulfone m-6 (2.0
equiv.) in an analogous manner as described for step SN-1. LCMS
data (ES+) M+Na 726.40. Triol m-16 (11 mg, 45%) was prepared in an
analogous manner as described for step SN-2.
[0825] The title Compound 168 (9.4 mg, 75%) was prepared from m-16
in an analogous manner as described for step SN-3. .sup.11H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.84-0.94 (m, 3H) 1.00 (s, 3H)
1.02 (s, 3H) 1.21 (s, 3H) 1.22-1.44 (m, 5H) 1.49-1.59 (m, 1H)
1.64-1.76 (m, 4H) 2.04-2.12 (m, 5H) 2.46-2.66 (m, 3H) 3.49-3.58 (m,
2H) 3.68-3.80 (m, 1H) 4.04-4.09 (m, 2H) 5.08 (d, J=8.8 Hz, 1H) 5.16
(d, J=11.2 Hz, 1H) 5.57-5.70 (m, 2H) 5.98-6.06 (m, 1H) 6.11 (d,
J=10.8 Hz, 1H) 6.25-6.34 (m, 1H) 6.53 (dd, J=8.4, 4.0 Hz, 1H) 6.67
(dd, J=7.2, 4.0 Hz, 1H) 7.46 (t, J=7.6 Hz, 1H). [0826] The sulfone
intermediate m-6 was prepared as described below.
##STR00336##
[0827] Step mm-1: A solution of isobutanol (1.2 equiv.) in DME (2
ml) was added to a suspension of potassium tert-butoxide (1.3
equiv.) in DME (3 ml, final conc. 0.41M) at room temperature. After
being stirred at 50.degree. C. for 30 minutes, bromopyridine m-1
(0.5 g, 2.05 mmol, 1.0 equiv.) was added to the mixture. After
being stirred at reflux for 2 hours, the reaction mixture was
cooled to room temperature, quenched with water and ethyl acetate
was added. The organic layer was washed with water, brine, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
crude product m-2 (0.49 g) was used in the next step without
further purification.
[0828] Step mm-2: To a solution of dioxolan m-2 (0.49 g, 2.05 mmol,
1.0 equiv.) in THF (0.27M) was added 5N hydrochloric acid (6.1
equiv.). After being stirred at room temperature for 3 hours, the
reaction mixture was quenched with 5N sodium hydroxide solution.
The aqueous layer was extracted with ethyl acetate and the organic
layer was washed with water, brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The crude product m-3 (0.39g)
was used in the next step without further purification.
[0829] Step mm-3: To a suspension of methyltriphenylphosphonium
iodide (1.3 equiv.) in THF (0.2M) at 0.degree. C. was added n-butyl
lithium (1.3 equiv., solution in n-hexane) dropwise. The reaction
was stirred at 0.degree. C. for 20 minutes. Next, a solution of
pyridine methyl ketone m-3 (0.39 g, 2 mmol, 1.0 equiv) in THF was
added dropwise. The reaction was stirred at 0.degree. C. for 30
minutes. The reaction was quenched with water and ethyl acetate was
added. The organic layer was washed with water and brine, dried
over magnesium sulfate, filtered, and concentrated in vacuo. The
crude product was purified by silica gel column chromatography
(heptane:ethyl acetate as eluant) to afford the desired product m-4
(0.35g, 91%). .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 1.01 (d,
J=6.8 Hz, 6H) 2.06-2.17 (m, 1H) 2.17 (s, 3H) 4.11 (d, J=6.4 Hz, 2H)
5.23 (s, 1H) 5.97 (s, 1H) 6.62 (d, J=8.0 Hz, 1H) 6.99 (d, J=7.6 Hz,
1H) 7.52 (dd, J=8.0, 7.6 Hz, 1H).
[0830] Step mm-4: To a solution of olefin m-4 (0.35 g, 1.82 mmol,
1.0 equiv.) in dichloromethane (0.18M) at 0.degree. C. was added
9-BBN (2.5 equiv., solution in hexane). The reaction was stirred at
50.degree. C. for 4.5 hours. After cooling to 0.degree. C., the
reaction was quenched with water, 5N sodium hydroxide solution (4.0
equiv.) and 30% aqueous hydrogen peroxide solution (4.0 equiv.).
After being stirred at room temperature for 1 hour, the mixture was
extracted with ethyl acetate. The organic layer was washed with
aqueous sodium thiosulfate solution, water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The crude
product was purified by silica gel column chromatography (heptane:
ethyl acetate as eluant) to afford the desired product m-5 (0.26g,
68%). LCMS data (ES+) M+Na 232.02.
[0831] The desired intermediate m-6 (315 mg, 63% in 2 steps) was
prepared in an analogous manner as described for Scheme 23. LCMS
data (ES+) M+Na 402.08. [0832] Synthesis of Compound 169
##STR00337##
[0833] The title compound 169 (0.30 mg, 0.8% in 3 steps) was
prepared from aldehyde sn-1 (40.0 mg, 0.076 mmol, 1.00 equiv.) and
nm-12 (1.81 equiv.) in an analogous manner as described for step
SN-1, SN-2 and SN-3. MS(ES+): 522.16 (M+Na.sup.+). [0834] The
sulfone intermediate nm-12 was prepared in the following
manner.
##STR00338##
[0835] Step NM-10: nm-13 (600 mg) was prepared from
2-methyl-3-buten-1-ol (200 mg, 2.32 mmol, 1.00 equiv.) in an
analogous manner as described in Scheme 23. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 1.16-1.81(m, 3H) 2.65-2.72(m, 1H)
3.40-3.43(m, 2H) 5.03-5.11(m, 2H) 5.71-5.80(m, 1H) 7.27-7.35(m, 1H)
7.51-7.59(m, 4H). Step NM-11: 9-BBN (3.00 equiv., 0.4 M solution in
THF) was added dropwise to a solution of nm-13 (300 mg, 1.22 mmol,
1.00 equiv.) in THF (2.5 mL) at 0.degree. C. under N.sub.2. The
reaction mixture was stirred at room temperature for 3 h. This
mixture was added to a solution of 2-bromopyridine (1.20 equiv.),
tetrakis(triphenylphosphine)palladium (0.20 equiv.) and potassium
carbonate (4.00 equiv.) in dimethylformamide (4.00 mL) and
distilled water (1.50 mL). The reaction mixture was stirred at
90.degree. C. for 4 hours under N.sub.2. The reaction mixture was
cooled to room temperature, filtered, extracted with ethyl acetate,
washed with water and brine, dried over magnesium sulfate,
filtered, and concentrated in vacuo. The residue was purified by
silica gel column chromatography (heptane/ethyl acetate as eluent)
to afford the desired product nm-14 along with a by-product (254
mg). The crude product nm-14 (254 mg) was used in the next step
without further purification.
[0836] Step NM-12: nm-12 (49.0 mg, 11% in 2 steps) was prepared
from nm-14 (254 mg, 0.78 mmol, 1.00 equiv.) in an analogous manner
as described for Scheme 23. MS(ES+): 379.90 (M+Na.sup.+). [0837]
Synthesis of Compound 170
##STR00339##
[0838] The title compound 170 (5.44 mg, 13% in 3 steps) was
prepared from aldehyde sn-1 (40.0 mg, 0.085 mmol, 1.00 equiv.) and
nm-15 (1.72 equiv.) in an analogous manner as described for step
SN-1, SN-2 and SN-3. .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.86-0.89 (m, 3H) 1.03 (d, J=6.40 Hz, 3H) 1.21 (s, 3H) 1.24-1.41
(m, 2H) 1.51-1.61 (m, 2H) 1.68 (s, 3H) 2.09 (s, 3H) 2.47-2.54 (m,
2H) 2.60-2.63 (m, 1H) 2.69-2.88 (m, 3H) 3.55 (d, J=10.8 Hz, 1H)
3.71-3.82 (m, 1H) 5.09 (d, J=9.2 Hz, 1H) 5.14 (d, J=10.8 Hz, 1H)
5.58-5.67 (m, 2H) 5.69-5.77 (m, 1H) 6.03-6.06 (m, 1H) 6.12-6.20 (m,
1H) 7.08-7.12 (m, 2H) 7.55-7.60 (m, 1H) 8.54-8.5 (m, 1H). MS(ES+):
508.07(M+Na+). [0839] The sulfone intermediate nm-15 was prepared
in the following manner.
##STR00340##
[0840] Step NM-13: nm-16 (2.84 g, 88%) was prepared from methallyl
alcohol (1.00 g, 13.9 mmol, 1.00 equiv.) in an analogous manner as
described for step #40-6. .sup.1H NMR (400 MHz, CHLOROFORM-d)
.delta.: 1.84 (s, 3H) 4.05 (s, 2H) 4.94-4.96 (m, 1H) 5.09-5.10 (m,
1H) 7.54-7.59 (m, 5H).
[0841] Step NM-14: nm-17 (150 mg, 56%) was prepared from nm-16 (200
mg, 0.861 mmol, 1.00 equiv.) in an analogous manner as described
for step NM-11. .sup.11-1 NMR (400 MHz, CHLOROFORM-d) .delta.: 1.01
(d, J=6.80 Hz, 3H) 2.52-2.57 (m, 1H) 2.72-2.78 (m, 1H) 2.97-3.02
(m, 1H) 3.37-3.42 (m, 1H) 3.49-3.54 (m, 1H) 7.11-7.16 (m, 2H)
7.52-7.66 (m, 6H) 8.52-8.54 (m, 1H).
[0842] Step NM-15: nm-15 (50.0 mg, 30%) was prepared from nm-17
(150 mg, 0.482 mmol, 1.00 equiv.) in an analogous manner as
described for Scheme 23. MS(ES+): 365.92 (M+Na.sup.+).
[0843] Synthesis of Compound 171
##STR00341##
[0844] The title Compound 171 (2.37 mg, 11.3%) was prepared from
diene x (15 mg, 0.041 mmol, 1.0 equiv.) and sn-5 (3.00 equiv.) in
an analogous manner as described for step MM-1. .sup.1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.91 (d, J=6.80 Hz, 3H) 1.22 (s, 3H)
1.37 (s, 3H) 1.22-1.58 (m, 4H) 1.68-1.74 (m, 1H) 1.78 (s, 3H)
1.81-1.90 (m, 1H) 2.08 (br. s., 1H), 2.10 (s, 3H) 2.50-2.57 (m,
2H), 2.59-2.69 (m, 3H) 3.52 (d, J=10.8 Hz, 1H), 3.73-3.76 (m, 1H)
5.09 (d, J=9.20 Hz, 1H) 5.18 (d, J=10.8 Hz, 1H) 5.59-5.71 (m, 1H)
5.86 (d, J=15.2 Hz, 1H) 6.13 (d, J=10.8 Hz, 1H) 6.47 (d, J=15.2 Hz,
1H) 6.49 (d, J=15.2 Hz, 1H) 7.18-7.19 (m, 2H) 7.25-7.29 (m, 3H).
MS(ES+): 537.2 (M+Na.sup.+). [0845] The intermediate allelic
alcohol nm-11 was prepared as described in following manner.
##STR00342##
[0846] Step NM-5: To a stirred solution of the triethyl
phosphonoacetate (1.30 equiv.) in THF (0.673 M) was added sodium
hydride (1.50 equiv., >60% purity) at 0.degree. C. under
N.sub.2. The reaction mixture was stirred at room temperature for
60 min. Benzylacetone (3.00 g, 20.2 mmol, 1.00 equiv.) was added to
the reaction mixture at room temperature, then the reaction mixture
was stirred at room temperature overnight. The reaction mixture was
diluted with water and ethyl acetate, the organic layer was washed
with brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (heptane/ethyl acetate as eluent) to afford
the desired product nm-7 (4.30 g, 98%). .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 1.26-1.29 (m, 3H) 2.21 (s, 3H) 2.42-2.46 (m,
2H) 2.76-2.81 (m, 4H) 4.12-4.17 (m, 2H) 5.69 (s, 3H) 7.16-7.31 (m,
5H).
[0847] Step NM-6: To a stirred solution of nm-7 (4.30 g, 19.7 mmol,
1.00 equiv.) in toluene (0.281 M) was added dropwise
diisobutylaluminum hydride (2.20 equiv., 1.02 M solution in
toluene) at -78.degree. C. under N2. The reaction mixture was
stirred at -78.degree. C. for 2.5 hours. The reaction mixture was
diluted with water, sat. potassium sodium tartrate, ethyl acetate,
and stirred at room temperature for 1 hour. The organic layer was
washed with brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (heptane/ethyl acetate as eluent) to afford
the desired product nm-8 (1.75 g, 50%). .sup.1H NMR (400 1 MHz,
CHLOROFORM-d) .delta.: 1.72 (s, 3H) 2.30-2.34 (m, 2H) 2.71-2.76 (m,
2H) 4.12-4.13 (m, 2H) 5.38-5.43 (m, 1H) 5.69 (s, 3H) 7.16-7.30 (m,
5H).
[0848] Step NM-7: To a solution of titanium isopropoxide (1.20
equiv.) and 4.ANG. molecular sieves (1.00 g) in dichloromethane (10
mL) was added (-)-diethyl-D-tartrate (1.50 equiv.) in
dichloromethane (2.0 mL) at -20.degree. C. under N.sub.2. The
reaction mixture was stirred at -20.degree. C. for 10 min. nm-8
(1.75 g, 9.93 mmol, 1.00 equiv.) in dichloromethane (2.0 mL) was
added to the reaction mixture at -20.degree. C. The reaction
mixture was cooled to -30.degree. C., and tert-butyl hydroperoxide
(2.00 equiv., 6.0 M solution in nonane) in dichloromethane (1.0 mL,
final conc. 0.66 M) was added to the reaction mixture. The reaction
mixture was stirred at -30.degree. C. for 60 min. A solution of
water (20 mL), iron sulfate heptahydrate (1.43 equiv.) and
D-(-)-tartaric acid (12.0 equiv.) was added to the reaction
mixture, then stirred at 0.degree. C. for 20 min. The reaction
mixture was diluted with ethyl acetate and further extracted with
ethyl acetate. A 1N NaOH aqueous solution (10 mL) was added to the
organic layer and the mixture was stirred at room temperature for
30 min. Then, water was added to the mixture. The organic layer was
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The residue was purified by silica gel
column chromatography (heptane/ethyl acetate as eluent) to afford
the mixture of desired product nm-9 and (-)-diethyl-D-tartrate. The
crude product nm-9 (1.90 g) was used in the next step without
further purification.
[0849] Step NM-8: A mixture of crude product nm-9 (1.90 g, 9.88
mmol, 1.00 equiv.), p-toluenesulfonyl chloride (2.00 equiv.) and
triethylamine (5.00 equiv.) in dichloromethane (15 mL, 0.659 M) was
stirred at room temperature for 60 min under N.sub.2. The reaction
mixture was diluted with water and ethyl acetate. The organic layer
was washed with brine, dried over magnesium sulfate, filtered, and
concentrated in vacuo. The residue was purified by silica gel
column chromatography (heptane/ethyl acetate as eluent) to afford
the desired product nm-10 with by-product (3.80 g). The crude
product nm-10 (3.80 g) was used in the next step without further
purification.
[0850] Step NM-9: Sodium iodide (4.00 equiv.) was added to a
mixture of crude product nm-10 (1.00 g, 2.89 mmol, 1.00 equiv.) in
THF (20 mL, 0.145 M) at room temperature. The reaction mixture was
stirred at 70.degree. C. for 60 min. After nm-10 was no longer
detected by TLC, zinc copper couple (5.00 equiv.) was added to the
reaction mixture. The reaction mixture was stirred at reflux for 3
hours. The reaction mixture was diluted with ethyl acetate,
filtered through Celite.RTM., washed with brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The residue
was purified by silica gel column chromatography (heptane/ethyl
acetate as eluent) to afford the desired product nm-11 (249 mg).
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 1.35 (s, 3H) 1.82-1.89
(m, 2H) 2.62-2.69 (m, 2H) 5.10-5.13 (m, 1H) 5.25-5.29 (m, 1H)
5.94-6.01 (m, 1H) 7.16-7.30 (m, 5H). [0851] Synthesis of Compound
172
##STR00343##
[0852] Step NM-1: To a stirred solution of nm-2 (50.0 mg, 0.159
mmol, 1.52 equiv.) in THF (2.00 mL) at -78.degree. C. under Na was
slowly added KHMDS (1.60 equiv., 0.50 M solution in toluene). The
reaction mixture was stirred at -78.degree. C. for 60 minutes.
Aldehyde nm-1 (50.0 mg, 0.104 mmol, 1.0 equiv.) in THF (1.00 mL,
final conc. 0.035 M) was added slowly at -78.degree. C. At the same
temperature, the reaction mixture was stirred for 60 min. The
reaction mixture was allowed to warm to room temperature. The
reaction mixture was diluted with water and ethyl acetate. The
aqueous layer was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The residue was
purified by silica gel column chromatography (heptane/ethyl acetate
as eluent) to afford the desired product nm-3 (32.0 mg, 54%).
[0853] Step NM-2: To a stirred solution of the nm-3 (18.0 mg, 0.032
mmol, 1.0 equiv.) in THF (0.032 M) was slowly added
tetrabutylammonium fluoride (2.00 equiv., 1.00 M solution in THF)
at room temperature under N.sub.2. The reaction mixture was stirred
at room temperature for 60 min. Tetrabutylammonium fluoride (2.00
equiv., 1.00 M solution in THF) was added to the reaction mixture,
and then the reaction was stirred at room temperature for 30 min.
The reaction mixture was diluted with water and ethyl acetate. The
aqueous layer was extracted with ethyl acetate. The combined
organic layers were washed with brine, dried over magnesium
sulfate, filtered, and concentrated in vacuo. The residue was
purified by preparative silica gel column chromatography (ethyl
acetate only) to afford the title Compound 172 (3.57 mg, 25%).
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.89 (d, J=6.80 Hz,
3H) 1.21 (s, 3H) 1.22-1.70 (m, 4H) 1.74 (s, 3H) 2.10 (s, 3H)
2.48-2.64 (m, 3H) 3.48-3.62 (m, 1H) 3.65 (d, J=6.80 Hz, 2H)
3.72-3.78 (m, 1H) 5.08 (d, J=8.80 Hz, 1H) 5.16 (d, J=10.40 Hz, 1H)
5.57-5.70 (m, 2H) 5.94-6.02 (m, 1H) 6.13 (d, J=11.2 Hz, 1H)
6.34-6.41 (m, 1H) 7.11-7.18 (m, 2H) 7.59-7.63 (m, 1H) 8.53-8.55 (m,
1H). MS(ES+): 480.18 [M+Na.sup.+].
[0854] The aldehyde intermediate nm-1 was prepared as previously
described (R. M. Kanada and D. Ito et. al., PCT Int. Appl, 2007,
WO2007043621) and sulfone intermediate nm-2 was prepared in the
following manner.
##STR00344##
[0855] Step NM-3: Sulfide nm-4 (2.03 g, 88%) was prepared from
2-(2-hydroxylethyl)-pyridine (1.00 g, 8.12 mmol, 1.00 equiv.) in an
analogous manner as described in Scheme 23. .sup.1H NMR (400 MHz,
CHLOROFORM-d) .delta.: 3.37-3.41 (m, 2H) 3.81-3.86 (m, 2H)
7.16-7.25 (m, 2H) 7.52-7.66 (m, 6H) 8.55-8.57 (m, 1H), MS(ES+):
305.98 [M+Na.sup.+].
[0856] Step NM-4: Sulfone nm-2 (132 mg, 40%) was prepared from nm-4
(300 mg, 1.06 mmol, 1.00 equiv.) in an analogous manner as
described in Scheme 23. 1H NMR (400 MHz, CHLOROFORM-d) .delta.:
3.46-3.50 (m, 2H) 4.25-4.29 (m, 2H) 7.16-7.24 (m, 2H) 7.58-7.72 (m,
6H) 8.49-8.50 (m, 1H), MS(ES+): 337.90 [M+Na.sup.+].
Synthesis of Compound 173
##STR00345##
[0858] The title compound 173 (0.40 mg, 1.4% in 2 steps) was
prepared from nm-1 (30 mg, 0.062 mmol, 1.00 equiv.) and nm-5 (2.55
equiv.) in an analogous manner as described for steps NM-1 and 2.
.sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.89 (d, J=6.80 Hz,
3H) 1.21 (s, 3H) 1.22-1.81 (m, 4H) 1.73 (s, 3H) 2.10 (s, 3H)
2.48-2.65 (m, 3H) 3.44-3.52 (m, 3H) 3.73-3.78 (m, 1H) 5.08 (d,
J=9.20 Hz, 1H) 5.16 (d, J=10.8 Hz, 1H) 5.58-5.71 (m, 2H) 5.82-5.89
(m, 1H) 6.10-6.13 (m, 1H) 6.28-6.34 (m, 1H) 7.11-7.13 (m, 2H)
8.50-8.52 (m, 2H). MS(ES+): 480.18 (M+Na.sup.+).
[0859] The sulfone intermediate nm-5 (357 mg, crude) was prepared
from 4-(2-hydroxyl-ethyl)pyridine (1.00 g, 8.12 mmol, 1.00 equiv.)
in an analogous manner as described for step NM-3 and NM-4. .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta.: 3.29-3.33 (m, 2H) 4.02-4.07
(m, 2H) 7.22-7.23 (m, 2H) 7.44-7.72 (m, 6H) 8.59-8.60 (m, 1H).
[0860] Synthesis of Compound 174
##STR00346##
[0861] The title Compound 174 (3.71 mg, 13.0% in 2 steps) was
prepared from nm-1 (30 mg, 0.062 mmol, 1.00 equiv.) and nm-6 (2.55
equiv.) in an analogous manner as described for steps NM-1 and
NM-2. .sup.1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.89 (d, J=6.80
Hz, 3H) 1.21 (s, 3H) 1.22-1.73 (m, 4H) 1.73 (s, 3H) 2.10 (s, 3H)
2.48-2.65 (m, 3H) 3.46 (d, J=6.80 Hz, 2H) 3.51 (d, J=11.2 Hz, 1H)
3.72-3.76 (m, 1H) 5.08 (d, J=8.80 Hz, 1H) 5.15 (d, J=10.8 Hz, 1H)
5.57-5.72 (m, 2H) 5.83-5.90 (m, 1H) 6.09-6.12 (m, 1H) 6.26-6.32 (m,
1H) 7.21-7.26 (m, 1H) 7.48-7.52 (m, 1H) 8.45-8.48 (m, 2H). MS(ES+):
480.18 (M+Na.sup.+).
[0862] The sulfone intermediate nm-6 (2.30 g, crude) was prepared
from 3-(2-hydroxylethyl)-pyridine (1.00 g, 8.12 mmol, 1.00 equiv.)
in an analogous manner as described for step NM-3 and NM-4. .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta.: 3.28-3.34 (m, 2H) 4.01-4.05
(m, 2H) 7.25-7.31 (m, 2H) 7.44-7.73 (m, 6H) 8.55-8.56 (m, 1H).
TABLE-US-00012 TABLE 9 Compounds 163-174 LCMS Structure, Compound
#, and Chemical Name .sup.1H NMR data data (ES+) ##STR00347## 1H
NMR (400 MHz, CHLOROFORM-d) .delta.: 0.90 (d, J = 6.4 Hz, 3 H) 1.21
(s, 3 H) 1.23- 1.47 (m, 5 H) 1.48-1.74 (m, 4 H) 1.77 (s, 3 H)
1.79-1.94 (m, 2 H) 2.10 (s, 3 H) 2.47-2.74 (m, 5 H) 3.54 (d, J =
10.4 Hz, 1 H) 3.75 (br. s., 1 H) 5.09 (d, J = 8.8 Hz, 1 H) 5.18 (d,
J = 10.8 Hz, 1 H) 5.58-5.72 (m, 2 H) 5.86 (d, J = 15.2 Hz, 1 H)
6.13 (d, J = 10.8 Hz, 1 H) 6.48 (dd, J = 15.2, 10.8 Hz, 1 H)
7.10-7.22 (m, 3 H) 7.23-7.31 (m, 2 H) 537.3 (M + Na.sup.+)
##STR00348## 1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.80- 0.95 (m,
3 H) 1.15-1.46 (m, 5 H) 1.60- 1.78 (m, 9 H) 1.90 (s, 1 H) 2.1 (s, 3
H) 2.45-2.67 (m, 3 H) 3.51 (d, J = 10.8 Hz, 1 H) 3.76 (br. s., 1 H)
5.09 (d, J = 8.8 Hz, 1 H) 5.17 (d, J = 10.4 Hz, 1 H) 5.57-5.71 (m,
2 H) 6.06 (d, J = 14.8 Hz, 1 H) 6.14 (d, J = 10.0 Hz, 1 H) 6.50
(ddd, J = 15.2, 10.8, 8.4 Hz, 1 H) 7.21-7.30 (m, 1 H) 7.31-7.39 (m,
2 H) 7.40-7.49 (m, 2 H) 509.3 (M + Na.sup.+) ##STR00349## 1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.82-0.98 (m, 3 H) 1.15-1.44 (m, 8
H) 1.47- 1.88 (m, 5 H) 1.99-2.26 (m, 5 H) 2.44-2.74 (m, 3 H)
3.44-3.61 (m, 1 H) 3.75 (br. s., 1 H) 5.02-5.26 (m, 2 H) 5.53-5.75
(m, 2 H) 6.00-6.22 (m, 2 H) 6.47-6.63 (m, 1 H) 6.89- 7.03 (m, 2 H)
7.17-7.35 (m, 1 H) 515.1 (M + Na.sup.+) ##STR00350## 1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.82- 0.95 (m, 3H), 1.21 (s, 3 H) 1.21-
1.45 (m, 5H) 1.48-1.49 (m, 2H) 1.63- 1.78 (m, 5 H) 2.09 (s, 3 H)
2.45- 2.68 (m, 3 H), 3.54 (q, J = 7.2 Hz, 1 H), 3.75 (br. s., 1 H)
5.08 (d, J = 9.2 Hz, 1 H) 5.15 (d, J = 10.4 Hz, 1 H) 5.57- 5.72 (m,
2 H) 5.93 (ddd, J = 15.2, 7.2, 2.4 Hz, 1 H) 6.10 (d, J = 10.4 Hz, 1
H) 6.17-6.28 (m, 1H), 7.16-7.32 (m, 5 H) 493.2 (M + Na.sup.+)
##STR00351## 1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.80- 0.95 (m,
3 H) 1.21 (s, 3 H) 1.24- 1.73 (m, 8 H) 1.74 (s, 3 H) 2.09 (s, 3 H)
2.42-2.67 (m, 3 H) 3.48-3.62 (m, 2 H) 3.74 (br. s., 1 H) 3.93 (s, 3
H) 5.08 (d, J = 8.40 Hz, 1 H) 5.16 (d, J = 10.40 Hz, 1 H) 5.57-5.70
(m, 2 H) 5.99-6.07 (m, 1 H) 6.11 (d, J = 10.80 Hz, 1 H) 6.25-6.34
(m, 1 H) 6.54 (d, J = 7.20 Hz, 1 H) 6.70 (d, J = 7.20 Hz, 1 H) 7.48
(t, J = 8.00 Hz, 1 H) 524.2 (M + Na.sup.+) ##STR00352## 1H NMR (400
MHz, CHLOROFORM-d) .delta.: 0.84- 0.94 (m, 3 H) 1.00 (s, 3 H) 1.02
(s, 3 H) 1.21 (s, 3 H) 1.22-1.44 (m, 5 H) 1.49-1.59 (m, 1 H)
1.64-1.76 (m, 4 H) 2.04-2.12 (m, 5 H) 2.46-2.66 (m, 3 H) 3.49-3.58
(m, 2 H) 3.68- 3.80 (m, 1 H) 4.04-4.09 (m, 2 H) 5.08 (d, J = 8.8
Hz, 1 H) 5.16 (d, J = 11.2 Hz, 1 H) 5.57-5.70 (m, 2 H) 5.98-6.06
(m, 1 H) 6.11 (d, J = 10.8 Hz, 1 H) 6.25-6.34 (m, 1 H) 6.53 (dd, J
= 8.4, 4.0 Hz, 1 H) 6.67 (dd, J = 7.2, 4.0 Hz, 1 H) 7.46 (t, J =
7.6 Hz, 1 H) ##STR00353## 522.16 (M + Na.sup.+) ##STR00354## 1H NMR
(400 MHz, CHLOROFORM-d) .delta.: 0.86- 0.89 (m, 3 H) 1.03 (d, J =
6.40 Hz, 3 H) 1.21 (s, 3 H) 1.24-1.41 (m, 2 H) 1.51- 1.61 (m, 2 H)
1.68 (s, 3 H) 2.09 (s, 3 H) 2.47-2.54 (m, 2 H) 2.60-2.63 (m, 1 H)
2.69-2.88 (m, 3 H) 3.55 (d, J = 10.8 Hz, 1 H) 3.71-3.82 (m, 1 H)
5.09 (d, J = 9.2 Hz, 1 H) 5.14 (d, J = 10.8 Hz, 1 H) 5.58-5.67 (m,
2 H) 5.69-5.77 (m, 1 H) 6.03-6.06 (m, 1 H) 6.12-6.20 (m, 1 H) 7.08-
7.12 (m, 2 H) 7.55-7.60 (m, 1 H) 8.54-8.55 (m, 1 H) 508.07 (M +
Na.sup.+) ##STR00355## 1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.91
(d, J = 6.80 Hz, 3 H) 1.22 (s, 3 H) 1.37 (s, 3 H) 1.22-1.58 (m, 4
H) 1.68- 1.74 (m, 1 H) 1.78 (s, 3 H) 1.81- 1.90 (m, 1 H) 2.08 (br.
s., 1 H), 2.10 (s, 3 H) 2.50-2.57 (m, 2 H), 2.59-2.69 (m, 3 H) 3.52
(d, J = 10.8 Hz, 1 H), 3.73-3.76 (m, 1 H) 5.09 (d, J = 9.20 Hz, 1
H) 5.18 (d, J = 10.8 Hz, 1 H) 5.59-5.71 (m, 1 H) 5.86 (d, J = 15.2
Hz, 1 H) 6.13 (d, J = 10.8 Hz, 1 H) 6.47 (d, J = 15.2 Hz, 1 H) 6.49
(d, J = 15.2 Hz, 1 H) 7.18-7.19 (m, 2 H) 7.25-7.29 (m, 3 H) 537.2
(M + Na.sup.+) ##STR00356## 1H NMR (400 MHz, CHLOROFORM-d) .delta.:
0.89 (d, J = 6.80 Hz, 3 H) 1.21 (s, 3 H) 1.22- 1.70 (m, 4 H) 1.74
(s, 3 H) 2.10 (s, 3 H) 2.48-2.64 (m, 3 H) 3.48-3.62 (m, 1 H) 3.65
(d, J = 6.80 Hz, 2 H) 3.72- 3.78 (m, 1H) 5.08 (d, J = 8.80 Hz, 1 H)
5.16 (d, J = 10.40 Hz, 1 H) 5.57- 5.70 (m, 2 H) 5.94-6.02 (m, 1 H)
6.13 (d, J = 11.2 Hz, 1 H) 6.34- 6.41 (m, 1 H) 7.11-7.18 (m, 2H)
7.59-7.63 (m, 1 H) 8.53-8.55 (m, 1 H) 480.18 (M + Na.sup.+)
##STR00357## 1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.89 (d, J =
6.80 Hz, 3 H) 1.21 (s, 3 H) 1.22- 1.81 (m, 4 H) 1.73 (s, 3 H) 2.10
(s, 3 H) 2.48-2.65 (m, 3H) 3.44-3.52 (m, 3 H) 3.73-3.78 (m, 1H)
5.08 (d, J = 9.20 Hz, 1 H) 5.16 (d, J = 10.8 Hz, 1 H) 5.58-5.71 (m,
2 H) 5.82- 5.89 (m, 1 H) 6.10-6.13 (m, 1 H) 6.28-6.34 (m, 1 H)
7.11-7.13 (m, 2H) 8.50-8.52 (m, 2 H) 480.18 (M + Na.sup.+)
##STR00358## 1H NMR (400 MHz, CHLOROFORM-d) .delta.: 0.89 (d, J =
6.80 Hz, 3 H) 1.21 (s, 3 H) 1.22- 1.73 (m, 4 H) 1.73 (s, 3 H) 2.10
(s, 3 H) 2.48-2.65 (m, 3 H) 3.46 (d, J = 6.80 Hz, 2 H) 3.51 (d, J =
11.2 Hz, 1 H) 3.72-3.76 (m, 1H) 5.08 (d, J = 8.80 Hz, 1 H) 5.15 (d,
J = 10.8 Hz, 1 H) 5.57-5.72 (m, 2 H) 5.83- 5.90 (m, 1 H) 6.09-6.12
(m, 1 H) 6.26-6.32 (m, 1 H) 7.21-7.26 (m, 1H) 7.48-7.52 (m, 1 H)
8.45- 8.48 (m, 2 H) 480.18 (M + Na.sup.+)
Protocol for Synthesis of Compound 175
##STR00359##
[0864] Compound 175 was synthesized in an analogous manner as
Compound 163. MS(ES+): 553.35 [M+Na].sup.+. [0865] Protocol for
Synthesis of Compound 176
##STR00360##
[0866] Compound 176 was prepared from aldehyde sn-1 (1 equiv) and
SPE-21 (1.81 equiv.) in an analogous manner as described for step
SN-1, SN-2 and SN-3. MS(ES+): 521.42 (M+Na.sup.+). [0867] Protocol
for Synthesis of Compound 177
##STR00361##
[0868] Step 1: To a suspension of sodium hydride (55% oil
dispersion in mineral oil, 10.7 mmol, 1 equiv) in DMF (10 mL) at
0.degree. C. was added a solution of SPE-22 (2.0 g, 10.7 mmol, 1
equiv) dropwise at and was stirred for 30 minutes. Then iodomethane
(2 ml, 32.1 mmol, 3 equiv) was added dropwise and then allowed to
warm up to room temperature over 4 hrs. The reaction was quenched
with saturated ammonium chloride solution and diluted with ethyl
acetate. The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product
(SPE-23, 997 mg, 4.96 mmol, 46%).
[0869] Step 2: To a solution of 2-methylbut-3-en-1-ol (651 mg, 3.24
mmol, 1 equiv) in THF (1.25 mL) was added 9-BBN (0.5 M THF
solution, 11.6 mL, 5.8 mmol, 1.8 equiv) at 0.degree. C. dropwise.
The mixture was then warmed up to room temperature and was stirred
for 2.5 hours. To the reaction mixture, was added pre-mixed
solution of SPE-23 (651 mg, 3.24 mmol),
tetrakis(triphenylphosphine)palladium(0) (189 mg, 0.232 mmol, 0.07)
and potassium carbonate (962 mg, 6.96 mmol, 2.14 equiv) in DMF (24
mL) and H.sub.2O (0.75 mL). The resulting mixture was warmed up to
90.degree. C. and was stirred for 4 hours. The reaction mixture was
diluted with ethyl acetate and water. Phase separated and organic
layer was washed with H.sub.2O and brine, then was dried over
MgSO.sub.4. Solid was filtered out and solvent was removed in
vacuo. The obtained residue was purified by silica gel
chromatography (Heptane/EtOAc= 75/25) to give a product (SPE-24,
368 mg, 1.77 mmol, 76% yield).
[0870] Step 3: To a solution of SPE-24 (368 mg, 1.77 mmol, 1 equiv)
in THF (4 mL) at 0.degree. C. was added
1-phenyl-1H-tetrazole-5-thiol (378 mg, 2.12 mmol, 1.2 equiv),
triphenyl phosphine (557 mg, 2.12 mmol, 1.2 equiv) and diisopropyl
azodicarboxylate (452 mg, 2.12 mmol, 1.2 equiv) and was stirred for
3 hours. The reaction was diluted with ethyl acetate and H.sub.2O.
The organic layer was washed with water, brine, dried over
magnesium sulfate, filtered, and concentrated in vacuo. The
resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product
(SPE-25, 310 mg, 0.841 mmol, 48%).
[0871] Step 4: To a solution of SPE-26 (310 mg, 0.84 mmol, 1 equiv)
in ethanol (3 mL) was added a solution of ammonium molybdate
tetrahydrate (104 mg, 0.084 mmol, 0.1 equiv) in hydrogen peroxide
(35% aqueous solution, 1 ml, 12.6 mmol, 15 equiv) at room
temperature The reaction mixture was stirred for 16 hours. The
reaction mixture was diluted with ethyl acetate and water. Phase
separated and organic layer was washed with H.sub.2O, saturated aq.
NaS.sub.2O.sub.3, and brine, then was dried over magnesium sulfate.
Solid was filtered out and solvent was removed in vacuo. The
obtained residue was purified by silica gel chromatography
(Heptane/AcOEt= 2/1) to afford the desired product (SPE-27, 236 mg,
0.589 mmol, 70% yield).
[0872] Step 5: To a solution of SPE-27 (0.035 g, 0.087 mmol, 1.4
equiv) in THF (3.0 mL) under nitrogen at -78.degree. C. was added
KHMDS (0.5 M in THF solution, 0.20 mL, 0.10 mmol, 1.6 equiv)
dropwise and the reaction was stirred for 1 hour. Then aldehyde D
(0.030 g, 0.062 mmol, 1.0 equiv) in THF (0.2 mL) was added
dropwise. The reaction was stirred at -78.degree. C. for 3 hours.
The reaction was quenched with ammonium chloride and diluted with
ethyl acetate. The organic layer was washed with water, brine,
dried over magnesium sulfate, filtered, and concentrated in vacuo.
The resulting oil was purified by silica gel column chromatography
(hexane/ethyl acetate as eluent) to afford the desired product
(SPE-27, 0.024 g, 0.037 mmol, 59%).
[0873] Step 6: To a solution of SPE-27 (24.0 mg, 0.037 mmol, 1.0
equiv.) in THF (2.0 mL, 0.02 M) at room temperature was added
tributylammonium fluoride (1.0 M THF solution, 1.1 ml, 1.1 mmol, 30
equiv.) The reaction was stirred for 1 hour. The reaction was
diluted with ethyl acetate and H.sub.2O. The organic layer was
washed with water, brine, dried over magnesium sulfate, filtered,
and concentrated in vacuo. The resulting oil was purified by silica
gel column chromatography (hepatnes/ethyl acetate as eluent) to
afford the desired product (Compound 177, 6.8 mg, 0.012 mmol, 34%).
.sup.1H NMR (400 MHz, CHLOROFORM, d) .delta.: 0.90 (d, J=6.8 Hz,
3H) 1.07 (d, J=6.8 Hz, 3H) 1.21 (s, 3H) 1.24-1.38 (m, 3H) 1.51-1.70
(m, 4H) 1.74 (s, 3H) 2.05 (s, 3H) 2.27 (quin, J=6.8 Hz, 1H)
2.49-2.66 (m, 5H) 3.38 (s, 3H) 3.55 (d, J=10.8 Hz, 2H) 3.73-3.78
(m, 1 H) 4.44 (s, 2H) 5.08-5.19 (m, 2H) 5.61-5.73 (m, 3H) 6.09-6.26
(m, 2H) 7.16-7.32 (m, 5 H). MS(ES+): 565.36 [M+Na].sup.+. [0874]
Protocol for Synthesis of Compound 178
##STR00362##
[0875] Compound 178 (12.3 mg, 36.4% in 2 steps) was prepared from
aldehyde D (30.0 mg, 0.062 mmol, 1.00 equiv.) and sulfone SPE-28
(1.34 equiv.) in an analogous manner as described in Compound 182.
.sup.1H NMR (400 MHz, CHLOROFORM, d) .delta.: 0.90 (d, J=6.8 Hz,
3H) 1.07 (d, J=6.8 Hz, 3H) 1.21 (s, 3H) 1.24-1.41 (m, 3H) 1.51-1.70
(m, 4H) 1.73 (s, 3H) 2.05 (s, 3H) 2.20-2.24 (m, 1H) 2.49-2.66 (m,
5H) 3.38 (s, 3H) 3.56 (d, J=10.8 Hz, 2H) 3.73-3.78 (m, 1 H) 4.42
(s, 2H) 5.08-5.18 (m, 2H) 5.63-5.70 (m, 3H) 6.07-6.22 (m, 2H) 7.15
(d, J=8.0 Hz, 2H) 7.24 (d, J=8.0 Hz, 2H). MS(ES+): 565.37
[M+Na].sup.+. [0876] Protocol for Synthesis of Compound 179
##STR00363##
[0877] Compound 179 (11.5 mg, 34.1% in 2 steps) was prepared from
aldehyde D (30.0 mg, 0.062 mmol, 1.00 equiv.) and sulfone SPE-29
(1.34 equiv.) in an analogous manner as described in Compound 177.
.sup.1H NMR (400 MHz, CHLOROFORM, d) .delta.: 0.90 (d, J=6.8 Hz,
3H) 1.05 (d, J=6.8 Hz, 3H) 1.21 (s, 3H) 1.24-1.42 (m, 3H) 1.52-1.72
(m, 4H) 1.74 (s, 3H) 2.10 (s, 3H) 2.20-2.26 (m, 1H) 2.50-2.66 (m,
5H) 3.38 (s, 3H) 3.56 (dd, J=10.8, 3.2 Hz, 2H) 3.72-3.78 (m, 1H)
4.43 (s, 2H) 5.08-5.18 (m, 2H) 5.59-5.71 (m, 3H) 6.08-6.23 (m, 2H)
7.09-7.27 (m, 5H). MS(ES+): 565.41 [M+Na].sup.+. [0878] Protocol
for Synthesis of Compound 180
##STR00364##
[0879] To a solution of Pladienolide D (136 mg, 0.246 mmol, 1
equiv) in dichloromethane (4 mL) at room temperature was added
Dess-Martin periodinane (209 mg, 0.492 mmol, 2.0 equiv.). The
resulting solution was stirred for 10 minutes, and then was diluted
with ethyl acetate. The organic layer was washed with water and
brine, then was dried over sodium sulfate, solid was filtered off,
and solvent was removed in vacuo. The obtained residue was purified
by NH-silica gel chromatography (heptane/ethyl acetate as eluent)
to afford desired product (Compound 180, 66.1 mg, 0.12 mmol, 49%
yield). MS(ES+): 571.36 [M+Na].sup.+. [0880] Protocol for Synthesis
of Compound 181
##STR00365## ##STR00366##
[0881] Step 1: E7107 (48.00 g, 66.8 mmol, 1 equiv) was dissolved in
DMF (96 mL) and then imidazole (31.8 g, 467 mmol, 7 equiv) was
added. Upon complete dissolution of imidazole, the mixture was
cooled to 3.degree. C. TBSC1 (30.2 g, 200 mmol, 3 equiv) was added
and stirring was continued at 3-5.degree. C. for 2 hours. The
mixture was allowed to warm up to room temperature (18-19.degree.
C.) and stirring continued for 22 hours. The mixture was diluted
with MTBE (192 mL) and cooled to 3.degree. C. Water (192 mL) was
added while maintaining T-internal below 15.degree. C. and
resultant mixture was transferred to a separation funnel. Water (96
mL) and MTBE (96 mL) were used to rinse the reactor. Rinsate was
also transferred to the separation funnel and mixed well. The org
layer was separated and set aside. Aqueous layer was extracted with
MTBE twice (288 mL.times.2). All the organic layers were combined,
sequentially washed with: (1) water (96 mL), (2) 30 wt % aqueous
NaCl (96 mL, 492.79 mmol) and partially concentrated to give 631 g
yellow solution (silylation crude mixture), 1.58 g aliquot of
which, corresponding to 1/400 of crude mixture, was subjected to
purification by silica gel chromatography (25-50% MTBE/heptane) to
give the desired product (SPE-13, 172 mg).
[0882] Step 2: Another 1.58 g aliquot of the crude SPE-13 was
concentrated, dissolved in acetone (1.6 mL) and diluted with water
(0.4 mL). NMO (0.078 g, 0.68 mmol) was added followed by 2.5 wt %
O.sub.SO.sub.4 solution in water (0.34 ml, 0.033 mmol). After
overnight stirring (16 hr), the mixture was diluted with toluene
(0.8 mL), cooled to 0.degree. C. and quenched with 20 wt % aqueous
sodium sulfite (0.8 g). The mixture was partially concentrated and
extracted with EtOAc twice (4 mL.times.2). All the organic layers
were combined, washed with 36 wt % aqueous NaCl (0.4 mL) and
concentrated. Crude product thus obtained was purified by Biotage
25M (EtOAc 100% and EtPAc--MeOh 19:1 v/v) to give the desired
product (SPE-14, 117 mg).
[0883] Step 3: SPE-14 (80 mg, 0.082 mmol, 1 equiv) was dissolved in
acetonitrile (1.6 mL) and treated with lead tetraacetate
(Pb(AcO).sub.4; 74 mg, 0.17 mmol, 2 equiv) at room temperature.
After 30 min, mixture was diluted with ethyl acetate (3.2 mL),
filtered and washed with a mixture of 20 wt % aqueous sodium
sulfite (Na.sub.2SO.sub.3, 0.3 g, 0.5 mmol, 7.3 equiv) and 9 wt %
aqueous sodium bicarbonate (0.3 g, 0.3 mmol, 3.6 equiv). The
organic layer was separated and set aside. The aqueous layer was
extracted with ethyl acetate (3.2 mL). All the organic layers were
combined, washed with 36 wt % aqueous sodium chloride (0.60 mL),
and concentrated. Brownish crude oil thus obtained was purified by
short SiO.sub.2 plug column (EtOAc 100% & EtOAc 100% &
EtOAc--MeOH 9:1 v/v) to give the desired product (SPE-15, 30
mg).
[0884] Step 4: (Methyl)triphenylphosphonium bromide (1.28 g, 3.59
mmol, 2.2 equiv) was suspended in THF (10.5 mL) and cooled to
--10.degree. C. 1 M potassium tert-butoxide solution in THF (3.2
mL, 3.2 mmol, 2 equiv) was added (T-internal reached --6.1.degree.
C.) and the resultant yellow mixture was stirred at -10.degree. C.
After 30 min, the mixture was cooled to below --70.degree. C. A
solution of SPE-15 (1.049 g, 1.62 mmol, 1 equiv) in THF (2.1 mL)
was added (T.ltoreq.-65.degree. C.). Additional THF (2.1 mL) was
used for rinse. Dry ice/acetone bath was replaced with dry
ice/acetonitrile bath to have the mixture warmed up to approx.
--45.degree. C. After 30 min, 28 wt % aqueous ammonium chloride (1
g) was added and the mixture was allowed to warm up to --10.degree.
C., diluted with toluene (31.5 mL) and water (2 mL). The organic
layer was separated, washed with 36 wt % aqueous sodium chloride (3
mL), concentrated and purified by Biotage Snap Ultra 100 g (0-100%
EtOAc/acetone) to give the desired product (SPE-16, 310 mg).
[0885] Step 5-6: SPE-16 (0.110 g, 0.17 mmol, 1 equiv) was dissolved
in 1,2-dichloroethane (2.3 mL). Acrolein dimethyl acetal (0.20 ml,
1.7 mmol, 10 equiv), benzoquinone (0.5 mg) and Hoveyda-Grubbs
2.sup.nd generation catalyst (14 mg, 0.017 mmol, 0.1 equiv) were
added. The resultant mixture was heated at 50.degree. C. Additional
reagents were charged at the following time points: 1 hr--acrolein
dimethyl acetal (0.20 ml, 1.7 mmol, 10 equiv), 2 hr--Acrolein
dimethyl acetal (0.20 ml, 1.7 mmol, 10 equiv), 3 hr--Hoveyda-Grubbs
2nd generation catalyst (14 mg, 0.017 mmol, 0.1 equiv) and acrolein
dimethyl acetal (0.20 ml, 1.7 mmol, 10 equiv). Heating was
continued for extra 5 hr and the mixture was let cool down to
ambient temp. The mixture was directly loaded on silica gel column
for purification (heptane-MTBE 1:1, heptane-EtOAc 9:1) to give
crude SPE-17. This was dissolved in dichloromethane (1 mL) and
treated with formic acid (0.1 mL) at room temperature for 10
minutes. 9 wt % aqueous sodium bicarbonate (3 g) was carefully
added and the mixture was extracted with ethyl acetate twice (4
mL.times.2). All the organic layers were combined, concentrated and
purified by silica gel chromatography (EtOAc 100% &
EtOAc-acetone 3:1) to give the desired product (SPE-18, 10 mg)
[0886] Step 7-8: sn-2 (10.8 mg, 0.033 mmol, 2 equiv) was dissolved
in THF (0.1 mL). DMF (0.025 mL) was added and the mixture was
cooled to --70.degree. C. 0.5 M solution of 1 M NaHMDS solution in
THF (0.037 ml, 0.037 mmol, 2.5 equiv) was added (<-65.degree.
C.). A solution of SPE-18 (0.010 g, 0.015 mmol, 1 equiv) in THF
(0.1 mL) was added. (<-60.degree. C.). THF (0.2 mL) was used for
rinse. After 30 min, dry ice/acetone bath was replaced with dry
ice/MeCN bath. The mixture was allowed to warm up to -45.degree. C.
to -50.degree. C. After 1 hr, the reaction was quenched with 28 wt
% aqueous ammonium chloride (0.1 g). The mixture was warmed up to
0.degree. C., and then diluted with ethyl acetate (6 mL) and water
(0.2 mL). The organic layer was separated, washed with 36 wt %
aqueous sodium chloride (0.3 mL), concentrated and purified by
silica gel chromatography (50-100% EtOAc/heptane) to give the
desired product (SPE-19, 10 mg). SPE-19 was dissolved in THF (0.3
mL) and treated with 1 M TBAF solution in THF (0.030 mL, 0.03 mmol)
at room temperature. After overnight stirring, the mixture was
concentrated and purified by Sift plug (MTBE 100% to MTBE-acetone
2:1) to give the desired product (Compound 181, 3 mg). .sup.1H NMR
(400 MHz, CDCl.sub.3) .quadrature..quadrature. 8.54 (1H, m), 7.60
(1H, m), 7.09-7.17 (2H, m), 6.22-6.36 (2H, m), 6.14 (1H, m), 5.99
(1H, dd, J=7 Hz and 15 Hz), 5.68 (1H, dd, J=10 Hz and 15 Hz), 5.59
(1H, dd, J=10 Hz and 15 Hz), 5.16 (1H, d, J=10 Hz), 5.01 (1H, d,
J=10 Hz), 3.6-3.8 (2H, m), 3.4-3.5 (5H, m), 2.4-2.6 (8H, m), 1.96
(1H, s), 1.2-1.8 (16H, m), 1.73 (3H, s), 1.44 (3H, d, J=7 Hz), 1.22
(3H, s), 0.87 (3H, d, J=7 Hz). [0887] Protocol for Synthesis of
Compound 182
##STR00367##
[0888] Step 1: To H3B-8800 (55 mg, 0.099 mmol, 1 equiv) in DCE (5
mL), mCPBA (17.08 mg, 0.099 mmol, 1.0 equiv) was added and stirred
for 1 hr. The reaction mixture was evaporated and purified by
preparative HPLC to give the desired product (Compound 182, 21 mg,
0.037 mmol, 37%). .sup.1H NMR (400 MHz, CDC13) .delta.: 0.80-1.00
(m, 3H) 1.23-1.48 (m, 6H) 1.50-1.63 (m, 1H) 1.65-1.83 (m, 4H)
2.41-2.68 (m, 5H) 3.19-3.36 (m, 7H) 3.67-3.85 (m, 2H) 3.91 (br s,
2H) 4.02 (br s, 2H), 5.03 (br d, J=9.54 Hz, 1H) 5.17 (d, J=10.54
Hz, 1H) 5.57-5.77 (m, 2H) 6.02 (dd, J=15.18, 7.40 Hz, 1H) 6.13 (br
d, J=11.04 Hz, 1H) 6.34 (dd, J=15.06, 10.79 Hz, 1H) 7.14 (t, J=6.18
Hz, 1H) 7.18 (d, J=7.14 Hz, 1H) 7.28 (s, 2H) 7.63 (td, J=7.65, 1.76
Hz, 1H) 8.56 (d, J=5.11 Hz, 1H). MS(ES+): 572.69 [M+H].sup.+.
[0889] Compounds 183 and 184 were synthesized according to Scheme
56.
##STR00368##
[0889] Exemplified Protocol for Synthesis of Compound 183
[0890] Step 1: To a solution of SPE-1 (246 mg, 0.746 mmol, 1.8
equiv) in a 1:2 ratio DMF (0.5 mL)/THF (1 mL) at -78.degree. C. was
added dropwise NaHMDS (0.829 mL, 0.829 mmol, 2.0 equiv) by slow
addition to ensure internal does not exceed -60.degree. C. The
yellow solution was stirred at -78.degree. C. for 30 mins. Then a
solution of D (200 mg, 0.414 mmol, 1 equiv) in THF (1 mL) was added
dropwise at such a rate to ensure the reaction temperature remained
below -60.degree. C. The flask was rinsed with additional THF (1
mL) and the reaction mixture stirred for 1 hr at -78. The bath temp
was increased to -50.degree. C. over 20 mins and then allowed to
stir between -50.degree. C. to -45.degree. C. for 2 hrs. Solid
ammonium chloride (22.16 mg, 0.414 mmol, 1 equiv) was added in one
portion. The bath was slowly allowed to warm to 0.degree. C. The
mixture was extracted with EtOAc, washed with brine, dried over
sodium sulfate, filtered, and concentrated. Purification of the
resulting residue by column chromatography (0-100% EtOAc/hexanes)
was completed to give the desired product (SPE-2, 320 mg, 0.382
mmol, 92%).
[0891] Step 2: To a solution of SPE-2 (320 mg, 0.382 mmol, 1 equiv)
in MeOH (3 mL) at rt was added solid potassium carbonate (74.0 mg,
0.535 mmol, 1.4 equiv) in one portion. The reaction mixture was
stirred at rt for 2.5 hrs. Then, it was cooled to 0.degree. C. and
solid ammonium chloride (28.6 mg, 0.535 mmol, 1 equiv) was added
along with water (2 mL). This mixture was extracted with EtOAc,
washed brine, dried over sodium sulfate, filtered, and
concentrated. The resulting residue was purified by silica gel
chromatography (0-100% EtOAc/hexanes) to give the desired product
(SPE-3, 148 mg, 0.272 mmol, 71%).
[0892] Step 3: To a 0.degree. C. solution of SPE-4 (17.87 mg, 0.069
mmol, 1.5) in DCM (1 mL) and Hunig'sBase (0.048 mL, 0.276 mmol, 6.0
equiv) was added phosgene (0.065 ml, 0.092 mmol, 2 equiv) (in
Toluene). The reaction mixture was stirred for 30 min at 0.degree.
C., then warmed to rt. The reaction mixture was concentrated by
rotavap and high vacuum. The residue was dissolved in THF (1 mL)
and SPE-3 (25 mg, 0.046 mmol, 1 equiv) and DMAP (22.47 mg, 0.184
mmol, 4 equiv) were added. The reaction mixture was stirred at rt
for 1 hr. The reaction mixture was then cooled to 0.degree. C. and
a 1M toluene solution of NaHMDS (0.184 ml, 0.184 mmol, 4 equiv) was
added. This was stirred for 2 hrs at this temperature. The reaction
mixture was quenched with ammonium chloride solution and extracted
with EtOAc, dried over sodium sulfate, filtered, and concentrated.
Purification by silica gel chromatography (10% MeOH/EtOAc) was
completed to give the desired product (SPE-5, 21 mg, 0.028 mmol,
60%).
[0893] Step 4: To SPE-5 (21 mg, 0.028 mmol, 1 equiv) in methanol
(0.5 mL), P-TOLUENESULFONIC ACID MONOHYDRATE (10.6 mg, 0.056 mmol,
2 equiv) was added at rt. After 3 hr, the reaction was quenched
with sat. NaHCO3 solution. The aqueous was extracted with EtOAc,
followed by washing with brine, driying over sodium sulfate,
filtering, and concentration to give the crude product.
Purification by silica gel chromatography (0-20% MeOH/EtOAc) was
completed to give the desired product (Compound 183, 15.7 mg, 0.024
mmol, 88%). .sup.1H NMR (400 MHz, METHANOL-d4) .delta.: ppm
0.86-0.92 (m, 3H) 1.14-1.18 (m, 1H) 1.21-1.24 (m, 3H) 1.27 (s, 2H)
1.36-1.48 (m, 7H) 1.57-1.69 (m, 2H) 1.76 (s, 3H) 1.81-1.98 (m, 6H)
2.50-2.62 (m, 6H) 2.72-2.87 (m, 4H) 3.62-3.70 (m, 1H) 3.75-3.84 (m,
1H) 4.91-4.97 (m, 1H) 5.02-5.10 (m, 1H) 5.52-5.64 (m, 1H) 5.67-5.79
(m, 1H) 5.89-6.00 (m, 1H) 6.10-6.19 (m, 1H) 6.32-6.42 (m, 1H)
7.35-7.45 (m, 1H) 7.70-7.81 (m, 1H) 8.37-8.41 (m, 1H) 8.42-8.45 (m,
1H). MS(ES+): 642.64 [M+H].sup.+. [0894] Protocol for the Synthesis
of Compound 185
##STR00369##
[0895] Step 1: To a solution of SPE-6 (184 mg, 0.559 mmol, 1.8
equiv) in 1:4 DMF (529 .mu.L)/THF (2139 .mu.L) at -78.degree. C.
was added dropwise 1M NaHMDS (482 .mu.L, 0.482 mmol, 1.55 equiv)
via slow addition to ensure internal does not exceed -60.degree. C.
The yellow solution was stirred at -78.degree. C. for 30 mins. Then
a solution of intermediate D (150 mg, 0.311 mmol, 1 equiv) in THF
(425 .mu.L) was added dropwise at such a rate as to ensure the
temperature remained below -60.degree. C. The aldehyde container
was rinsed with additional THF and added to main flask. The
reaction mixture was stirred for 1 hr, maintaining bath temp
between -70.degree. C. to -60.degree. C. The bath temperature was
increased to -50.degree. C. over 20 mins. This was then allowed to
stir between -50.degree. C. to -45.degree. C. for 2 hrs in an
acetonitrile-dry ice bath. After 2 hrs, solid AMMONIUM CHLORIDE
(72.6 mg, 1.358 mmol, 4.37 equiv) was added in one portion and bath
was allowed to slowly warm to 0.degree. C. Added toluene and water
at 0.degree. C. and the combined organics were washed with brine.
The organics were dried over sodium sulfate, filtered, and
concentrated. Purification by column chromatography (0-40%
MTBE/hexanes with long hold at 40% gave desired product as a
mixture with some aldehyde D (SPE-7, 57.8 mg, 0.099 mmol,
31.7%).
[0896] Step 2: To a solution of SPE-7 (29.2 mg, 0.05 mmol, 1 equiv)
in MeOH (252 .mu.L) at rt was added solid potassium carbonate (9.64
mg, 0.07 mmol, 1.4 equiv) in one portion. At 2 hours, the reaction
mixture was cooled to 0.degree. C. and sat aq ammonium chloride was
added. The aqueous was extracted with EtOAc and washed with brine.
The organics were dried over sodium sulfate, filtered, and
concentrated. The crude product (SPE-8, 12.6 mg, 0.023 mmol, 46.5%)
was taken into the next step without further purification.
[0897] Step 3: SPE-8 (24.2 mg, 0.045 mmol, 1 equiv) was dissolved
in methanol (225 .mu.L) and tosic acid (16.93 mg, 0.089 mmol, 2
equiv) was added. The reaction mixture was stirred at rt for 1 hr.
The reaction was quenched with sat. aq. sodium bicarbonate and
extracted with 10% MeOH/DCM. The combined organics were dried over
sodium sulfate, filtered, and concentrated. Purification by column
chromatography (0-20% MeOH/DCM) was completed to give the desired
product (Compound 185, 9.2 mg, 0.021 mmol, 48.1% yield) as a crusty
oil/white solid. 1H NMR (400 MHz, METHANOL-d4) .delta.: ppm 0.90
(d, J=6.78 Hz, 3H) 1.28 (s, 4H) 1.35-1.39 (m, 2H) 1.45 (d, J=7.03
Hz, 3H) 1.53-1.62 (m, 2H) 1.76 (d, J=0.88 Hz, 3H) 2.53 (s, 3H)
3.69-3.80 (m, 4H) 5.03-5.08 (m, 1H) 5.34-5.44 (m, 1H) 5.48-5.53 (m,
1H) 5.67-5.78 (m, 1H) 5.92-6.03 (m, 1H) 6.09-6.18 (m, 1H) 6.33-6.44
(m, 1H) 7.23-7.32 (m, 1H) 7.32-7.39 (m, 1H) 7.72-7.84 (m, 1H)
8.40-8.50 (m, 1H). MS(ES+): 430.43 [M+H].sup.+.
TABLE-US-00013 TABLE 10 Compounds 175-185 Structure, Compound #,
and Chemical LCMS data Name .sup.1H NMR data (ES+) ##STR00370##
553.35 ##STR00371## 521.42 ##STR00372## .sup.1H NMR (400 MHz,
CHLOROFORM, d) .delta.: 0.90 (d, J = 6.8 Hz, 3 H) 1.07 (d, J = 6.8
Hz, 3 H) 1.21 (s, 3 H) 1.24-1.38 (m, 3 H) 1.51-1.70 (m, 4 H) 1.74
(s, 3 H) 2.05 (s, 3 H) 2.27 (quin, J = 6.8 Hz, 1 H) 2.49-2.66 (m, 5
H) 3.38 (s, 3H) 3.55 (d, J = 10.8 Hz, 2 H) 3.73-3.78 (m, 1 H) 4.44
(s, 2 H) 5.08-5.19 (m, 2 H) 5.61-5.73 (m, 3 H) 6.09- 6.26 (m, 2 H)
7.16-7.32 (m, 5 H) 565.36 ##STR00373## .sup.1H NMR (400 MHz,
CHLOROFORM, d) .delta.: 0.90 (d, J = 6.8 Hz, 3 H) 1.07 (d, J = 6.8
Hz, 3 H) 1.21 (s, 3 H) 1.24-1.41 (m, 3 H) 1.51-1.70 (m, 4 H) 1.73
(s, 3 H) 2.05 (s, 3 H) 2.20-2.24 (m, 1 H) 2.49-2.66 (m, 5 H) 3.38
(s, 3 H) 3.56 (d, J = 10.8 Hz, 2 H) 3.73-3.78 (m, 1 H) 4.42 (s, 2
H) 5.08-5.18 (m, 2 H) 5.63-5.70 (m, 3 H) 6.07- 6.22 (m, 2 H) 7.15
(d, J = 8.0 Hz, 2 H) 7.24 (d, J = 8.0 Hz, 2 H) 565.37 ##STR00374##
.sup.1H NMR (400 MHz, CHLOROFORM, d) .delta.: 0.90 (d, J = 6.8 Hz,
3 H) 1.05 (d, J = 6.8 Hz, 3 H) 1.21 (s, 3 H) 1.24-1.42 (m, 3 H)
1.52-1.72 (m, 4 H) 1.74 (s, 3 H) 2.10 (s, 3 H) 2.20-2.26 (m, 1 H)
2.50-2.66 (m, 5 H) 3.38 (s, 3H) 3.56 (dd, J = 10.8, 3.2 Hz, 2 H)
3.72- 3.78 (m, 1 H) 4.43 (s, 2 H) 5.08- 5.18 (m, 2 H) 5.59-5.71 (m,
3 H) 6.08-6.23 (m, 2 H) 7.09-7.27 (m, 5 H) 565.41 ##STR00375##
571.36 ##STR00376## .sup.1H NMR (400 MHz, CDCl.sub.3) .delta.: 8.54
(1H, m), 7.60 (1H, m), 7.09-7.17 (2H, m), 6.22-6.36 (2H, m), 6.14
(1H, m), 5.99 (1H, dd, J = 7 Hz and 15 Hz), 5.68 (1H, dd, J = 10 Hz
and 15 Hz), 5.59 (1H, dd, J = 10 Hz and 15 Hz), 5.16 (1H, d, J = 10
Hz), 5.01 (1H, d, J = 10 Hz), 3.6-3.8 (2H, m), 3.4-3.5 (5H, m),
2.4-2.6 (8H, m), 1.96 (1H, s), 1.2-1.8 (16H, m), 1.73 (3H, s), 1.44
(3H, d, J = 7 Hz), 1.22 (3H, s), 0.87 (3H, d, J = 7 Hz)
##STR00377## .sup.1H NMR (400 MHz, CDCl3) .delta.: 0.80- 1.00 (m, 3
H) 1.23-1.48 (m, 6 H) 1.50-1.63 (m, 1 H) 1.65-1.83 (m, 4 H)
2.41-2.68 (m, 5 H) 3.19-3.36 (m, 7 H) 3.67-3.85 (m, 2 H) 3.91 (br
s, 2 H) 4.02 (br s, 2 H), 5.03 (br d, J = 9.54 Hz, 1 H) 5.17 (d, J
= 10.54 Hz, 1 H) 5.57-5.77 (m, 2 H) 6.02 (dd, J = 15.18, 7.40 Hz, 1
H) 6.13 (br d, J = 11.04 Hz, 1 H) 6.34 (dd, J = 15.06, 10.79 Hz, 1
H) 7.14 (t, J = 6.18 Hz, 1 H) 7.18 (d, J = 7.14 Hz, 1 H) 7.28 (s, 2
H) 7.63 (td, J = 7.65, 1.76 Hz, 1 H) 8.56 (d, J = 5.11 Hz, 1 H)
572.69 ##STR00378## .sup.1H NMR (400 MHz, METHANOL- d4) .delta.:
ppm 0.86-0.92 (m, 3 H) 1.14- 1.18 (m, 1 H) 1.21-1.24 (m, 3 H) 1.27
(s, 2 H) 1.36-1.48 (m, 7 H) 1.57-1.69 (m, 2 H) 1.76 (s, 3 H)
1.81-1.98 (m, 6 H) 2.50-2.62 (m, 6 H) 2.72-2.87 (m, 4 H) 3.62-3.70
(m, 1 H) 3.75-3.84 (m, 1 H) 4.91- 4.97 (m, 1 H) 5.02-5.10 (m, 1 H)
5.52-5.64 (m, 1 H) 5.67-5.79 (m, 1 H) 5.89-6.00 (m, 1 H) 6.10-6.19
(m, 1 H) 6.32-6.42 (m, 1 H) 7.35- 7.45 (m, 1 H) 7.70-7.81 (m, 1 H)
8.37-8.41 (m, 1 H) 8.42-8.45 (m, 1 H) 642.64 ##STR00379## .sup.1H
NMR (400 MHz, CHLOROFORM-d) .delta.: ppm 0.89- 0.93 (m, 3 H) 1.25
(s, 3 H) 1.26- 1.30 (m, 3 H) 1.32-1.51 (m, 1 H) 1.32-1.41 (m, 1 H)
1.43 (dd, J = 7.03, 2.26 Hz, 4 H) 1.49-1.58 (m, 1 H) 1.62-1.85 (m,
9 H) 1.93- 2.06 (m, 5 H) 2.17-2.22 (m, 2 H) 2.49-2.68 (m, 9 H)
2.73-2.88 (m, 2 H) 3.45-3.54 (m, 1 H) 3.55-3.63 (m, 1 H) 3.70-3.81
(m, 1 H) 4.17- 4.27 (m, 1 H) 4.98-5.04 (m, 1 H) 5.17 (d, J = 10.67
Hz, 1 H) 5.55- 5.66 (m, 1 H) 5.67-5.78 (m, 1 H) 5.86-5.97 (m, 1 H)
6.12 (d, J = 10.79 Hz, 1 H) 6.19-6.32 (m, 1 H) 7.22-7.27 (m, 1 H)
7.47-7.58 (m, 1 H) 8.44-8.54 (m, 1 H) 660.53 ##STR00380## .sup.1H
NMR (400 MHz, METHANOL- d4) .delta.: ppm 0.90 (d, J = 6.78 Hz, 3 H)
1.28 (s, 4 H) 1.35-1.39 (m, 2 H) 1.45 (d, J = 7.03 Hz, 3 H)
1.53-1.62 (m, 2 H) 1.76 (d, J = 0.88 Hz, 3 H) 2.53 (s, 3 H)
3.69-3.80 (m, 4 H) 5.03-5.08 (m, 1 H) 5.34-5.44 (m, 1 H) 5.48-5.53
(m, 1 H) 5.67-5.78 (m, 1 H) 5.92-6.03 (m, 1 H) 6.09- 6.18 (m, 1 H)
6.33-6.44 (m, 1 H) 7.23-7.32 (m, 1 H) 7.32-7.39 (m, 1 H) 7.72-7.84
(m, 1 H) 8.40-8.50 (m, 1 H) 430.43
Compounds 186-196 were synthesized according to Scheme 58. [0898]
Protocol for the synthesis of Compound 186
##STR00381##
[0899] Step 1: To a solution of tri-TES Pladienolide D (160 mg,
0.179 mmol) in 1,2-dichloroethane (5 mL) at 20.degree. C. was added
DMAP (32.7 mg, 0.268 mmol), triethyl amine (0.75 mL, 5.36 mmol) and
4-nitrophenyl chloroformate (360 mg, 1.787 mmol). The reaction
mixture was stirred at 40.degree. C. for 4 days, and at 60.degree.
C. for 2 hours. The reaction mixture was diluted with EtOAc and
washed with water, then the layers were separated. The aqueous
layer was extracted with EtOAc (2.times.). The combined organic
extracts were successively washed with water and brine, dried over
MgSO.sub.4, filtered, and concentrated in vacuo. Flash
chromatography afforded
(2S,3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-4R,2E,4E)-6-methyl-6-((triet-
hylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilypoxy)pentan-2-yl)oxira-
n-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilypoxy)oxacyclododec-4-e-
n-6-yl piperazine-1-carboxylate (150 mg, 79% yield).
[0900] .sup.1H -NMR (400 MHz, CHCl.sub.3-d): .delta. ppm 0.48-0.71
(m, 24H) 0.78-0.85 (m, 7H) 0.86-0.93 (m, 5H) 0.94-1.03 (m, 34H)
1.18-1.22 (m, 2H) 1.22-1.26 (m, 2H) 1.35-1.43 (m, 4H) 1.43-1.52 (m,
4H) 1.54 (s, 4H) 1.56-1.65 (m, 3H) 1.68-1.72 (m, 3H) 1.75 (br d,
J=0.75 Hz, 2H) 1.84-1.95 (m, 1H) 2.01-2.06 (m, 2H) 2.09 (s, 2H)
2.11 (s, 2H) 2.33-2.52 (m, 4H) 2.57 (dd, J=8.09, 2.07 Hz, 2H)
2.80-2.90 (m, 1H) 3.66-3.80 (m, 1H) 3.82-3.93 (m, 2H) 4.92-5.13 (m,
2H) 5.63-5.68 (m, 1H) 5.69-5.74 (m, 1H) 5.75-5.83 (m, 2H) 6.12 (br
d, J=10.67 Hz, 1H) 6.41 (ddd, J=15.15, 11.01, 5.08 Hz, 1H) 7.50 (d,
J=9.41 Hz, 2H) 8.35 (d, J=9.29 Hz, 2H).
[0901] Step 2: To a solution of
(2S,3S,6S,7R,10R,E)-3,7-dimethyl-2-4R,2E,4E)-6-methyl-6-((triethylsilyl)o-
xy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)he-
pta-2,4-dien-2-yl)-6-(((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethyls-
ilyl)oxy)oxacyclododec-4-en-7-yl acetate in DCM (1 mL) was added
piperazine (0.447 g, 5.195 mmol) and Hunig's base (0.9 mL, 5.195
mmol). The resulting yellowish suspension was stirred for 6 hours.
Reaction mixture was concentrated and chromatographed over silica
gel to afford
(2S,3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((trie-
thylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxi-
ran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec--
4-en-6-yl piperazine-1-carboxylate (1.0 g, 0.844 mmol, 81% yield).
LC/MS (ESI, m/z), 1008.1 [M+H].sup.+.
[0902] Step 3: 2S,
3S,6S,7R,10R,E)-7-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethyl-
silyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran--
2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-
-6-yl piperazine-1-carboxylate (1.09 g, 0.92 mmol), DCM (20.71 mL,
321.826 mmol), and DIPEA (19.91 mL, 114.018 mmol) were combined and
cooled to -78.degree. C. Hydrogen fluoride-pyridine (0.518 g, 5.232
mmol) was added and the reaction allowed to warm to RT and stirred
overnight. LC/MS suggested de-silylation. the reaction mixture was
cooled in an icebath. Saturated NaHCO.sub.3 was added and stirred
and extracted with DCM. The organic layers were combined, dried
over an. NA.sub.2SO.sub.4 and concentrated and chromatographed to
afford
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2S,3S-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
piperazine-1-carboxylate (225 mg, 36.8%). LC/MS (ESI, m/z), 665.6
[M+H].sup.+.
[0903] .sup.1H-NMR (400 MHz, CHCl.sub.3-d): .delta. ppm 0.87-0.92
(m, 6H) 0.94 (t, J=7.40 Hz, 3H) 1.16-1.31 (m, 1H) 1.35 (s, 3H)
1.40-1.56 (m, 4H) 1.59 (s, 3H) 1.66 (br dd, J=14.68, 7.03 Hz, 3H)
1.76-1.80 (m, 3H) 1.87 (dd, J=14.12, 5.46 Hz, 1H) 2.05 (s, 3H)
2.30-2.41 (m, 1H) 2.50 (d, J=3.76 Hz, 2H) 2.56-2.72 (m, 2H) 2.90
(br d, J=2.01 Hz, 1H) 3.19 (br t, J=5.14 Hz, 4H) 3.50-3.59 (m, 1H)
3.71 (br s, 4H) 3.77-3.89 (m, 1H) 5.01-5.13 (m, 2H) 5.58-5.71 (m,
1H) 5.71-5.81 (m, 1H) 5.88 (d, J=15.31 Hz, 1H) 6.15 (br d, J=10.79
Hz, 1H) 6.53 (dd, J=15.18, 10.92 Hz, 1H). [0904] Protocol for
synthesizing Compound 187
##STR00382##
[0905] Step 1: To a solution of tri-TES-Pladienolide D (200 mg,
0.223 mmol) in dichloromethane (2 mL) at 0.degree. C. was added
DMAP (409 mg, 3.35 mmol) and 4-nitrophenyl chloroformate (338 mg,
1.675 mmol). The reaction mixture was stirred at RT for 7 days,
diluted with EtOAc and water, then the layers were separated. The
aqueous layer was extracted with EtOAc (2.times.), and the combined
organic extracts were washed with brine. The combined organic
layers were dried over Na.sub.2SO.sub.4, filtered and concentrated
in vacuo. Flash chromatography afforded
(2S,3S,6S,7R,10R,E)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsilyl)-
oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)h-
epta-2,4-dien-2-yl)-7-(((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethyl-
silyl)oxy)oxacyclododec-4-en-6-yl acetate. (170 mg, 72% yield).
[0906] .sup.1H-NMR (400 MHz, CHCl.sub.3-d): .delta. ppm 0.54-0.67
(m, 18H) 0.78-1.03 (m, 36H) 1.19-1.32 (m, 1H) 1.39 (s, 3H)
1.43-1.52 (m, 3H) 1.55-1.63 (m, 3H) 1.64 (s, 3H) 1.74 (s, 3H) 1.88
(dd, J=13.80, 5.02 Hz, 1H) 2.13 (s, 3H) 2.23-2.37 (m, 1H) 2.39-2.48
(m, 2H) 2.51-2.63 (m, 2H) 2.84 (s, 1H) 3.69-3.77 (m, 1H) 3.82-4.00
(m, 1H) 5.04 (d, J=10.79 Hz, 1H) 5.24 (d, J=9.03 Hz, 1H) 5.67-5.84
(m, 3H) 6.12 (d, J=10.16 Hz, 1H) 6.42 (dd, J=15.06, 11.04 Hz, 1H)
7.42 (d, J=9.29 Hz, 2H) 8.29 (d, J=9.16 Hz, 2H).
[0907] Step 2: To a solution of
(2S,3S,6S,7R,10R,E)-3,7-dimethyl-2-4R,2E,4E)-6-methyl-6-((triethylsilyl)o-
xy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2-yl)he-
pta-2,4-dien-2-yl)-7-((4-nitrophenoxy)carbonyl)oxy)-12-oxo-10-((triethylsi-
lypoxy)oxacyclododec-4-en-6-yl acetate (100 mg, 0.094 mmol) in DCM
was added piperazine and DMAP. The resulting yellowish suspension
was stirred for 6 hours. The reaction mixture was concentrated to
give the crude product. Flash chromatography afforded
(2S,3S,6S,7R,10R,E)-6-acetoxy-3,7-dimethyl-2-4R,2E,4E)-6-methyl-6-((triet-
hylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxir-
an-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-
-en-7-yl piperazine-1-carboxylate (95 mg, 100%). LC/MS (ESI, m/z),
1008.8 [M+H].sup.+.
[0908] .sup.1-H-NMR (400 MHz, CHCl.sub.3-d): .delta. ppm 0.42-0.70
(m, 22H) 0.79-0.84 (m, 7H) 0.86-0.91 (m, 4H) 0.92-1.03 (m, 30H)
1.15-1.30 (m, 2H) 1.37-1.42 (m, 3H) 1.44-1.52 (m, 3H) 1.56-1.62 (m,
2H) 1.62-1.68 (m, 1H) 1.71-1.76 (m, 3H) 1.83-1.93 (m, 1H) 2.03-2.11
(m, 4H) 2.36-2.45 (m, 2H) 2.45-2.53 (m, 2H) 2.54-2.64 (m, 1H)
2.78-2.86 (m, 1H) 2.86-3.07 (m, 4H) 3.32-3.45 (m, 1H) 3.45-3.64 (m,
3H) 3.69-3.78 (m, 1H) 3.79-3.94 (m, 1H) 5.00 (d, J=10.54 Hz, 1H)
5.18 (s, 1H) 5.54-5.79 (m, 3H) 5.98-6.21 (m, 1H) 6.33-6.57 (m, 1H)
6.84-6.96 (m, 3H) 8.02-8.35 (m, 2H) 8.06-8.08 (m, 1H).
[0909] Step 3: To a solution of
(2S,3S,6S,7R,10R,E)-6-acetoxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((trie-
thylsilyl)oxy)-7-((2R,3R)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxi-
ran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec--
4-en-7-yl piperazine-1-carboxylate in (95 mg, 0.094 mmol) in THF (3
mL) was added TBAF (0.424 mL, 1 M, 0.424 mmol) and stirred at RT
for 10 hours. The mixture as concentrated and diluted with EtOAc,
washed with water and brine. The organic layer was separated and
dried with Na.sub.2SO.sub.4, filtered and concentrated in vacuo.
HPLC purification afforded
(2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy--
7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4--
dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-7-yl
piperazine-1-carboxylate (16 mg, 26%). LC/MS (ESI, m/z), 665.6
[M+H].sup.+.
[0910] .sup.1-H-NMR (400 MHz, CHCl.sub.3-d): .delta. ppm 0.90 (dd,
J=6.84, 2.20 Hz, 6H) 0.94 (t, J=7.40 Hz, 3H) 1.20-1.30 (m, 1H) 1.34
(s, 3H) 1.39-1.54 (m, 3H) 1.55 (s, 3H) 1.59-1.73 (m, 3H) 1.78 (d,
J=0.88 Hz, 3H) 1.86 (dd, J=13.99, 5.46 Hz, 1H) 2.05 (s, 3H)
2.39-2.53 (m, 3H) 2.55-2.65 (m, 1H) 2.67 (dd, J=8.03, 2.26 Hz, 1H)
2.89 (s, 1H) 3.22 (br s, 4H) 3.50-3.57 (m, 1H) 3.58-3.90 (m, 5H)
5.08 (d, J=10.67 Hz, 1H) 5.18 (d, J=9.03 Hz, 1H) 5.58-5.78 (m, 2H)
5.88 (d, J=15.31 Hz, 1H) 6.10-6.23 (m, 1H) 6.53 (dd, J=15.25, 10.98
Hz, 1H).
TABLE-US-00014 TABLE 11 Compounds 175-185 LCMS data Structure,
Compound #, and Chemical Name .sup.1H NMR data (ES+) ##STR00383##
.sup.1H-NMR (400 MHz, CHCl.sub.3-d): .delta. ppm 0.87-0.92 (m, 6 H)
0.94 (t, J = 7.40 Hz, 3 H) 1.16-1.31 (m, 1 H) 1.35 (s, 3 H)
1.40-1.56 (m, 4 H) 1.59 (s, 3 H) 1.66 (br dd, J = 14.68, 7.03 Hz, 3
H) 1.76-1.80 (m, 3 H) 1.87 (dd, J = 14.12, 5.46 Hz, 1 H) 2.05 (s, 3
H) 2.30-2.41 (m, 1 H) 2.50 (d, J = 3.76 Hz, 2 H) 2.56-2.72 (m, 2 H)
2.90 (br d, J = 2.01 Hz, 1 H) 3.19 (br t, J = 5.14 Hz, 4 H) 3.50-
3.59 (m, 1 H) 3.71 (br s, 4 H) 3.77- 3.89 (m, 1 H) 5.01-5.13 (m, 2
H) 5.58-5.71 (m, 1 H) 5.71-5.81 (m, 1 H) 5.88 (d, J = 15.31 Hz, 1
H) 6.15 (br d, J = 10.79 Hz, 1 H) 6.53 (dd, J = 15.18, 10.92 Hz, 1
H) LC/MS (ESI, m/z), 665.6 [M + H].sup.+ ##STR00384## .sup.1H-NMR
(400 MHz, CHCl.sub.3-d): .delta. ppm 0.90 (dd, J = 6.84, 2.20 Hz, 6
H) 0.94 (t, J = 7.40 Hz, 3 H) 1.20-1.30 (m, 1 H) 1.34 (s, 3 H)
1.39-1.54 (m, 3 H) 1.55 (s, 3 H) 1.59-1.73 (m, 3 H) 1.78 (d, J =
0.88 Hz, 3 H) 1.86 (dd, J = 13.99, 5.46 Hz, 1 H) 2.05 (s, 3 H)
2.39-2.53 (m, 3 H) 2.55- 2.65 (m, 1 H) 2.67 (dd, J = 8.03, 2.26 Hz,
1 H) 2.89 (s, 1 H) 3.22 (br s, 4 H) 3.50-3.57 (m, 1 H) 3.58-3.90
(m, 5 H) 5.08 (d, J = 10.67 Hz, 1 H) 5.18 (d, J = 9.03 Hz, 1 H)
5.58-5.78 (m, 2 H) 5.88 (d, J = 15.31 Hz, 1 H) 6.10- 6.23 (m, 1 H)
6.53 (dd, J = 15.25, 10.98 Hz, 1 H) LC/MS (ESI, m/z), 665.6 [M +
H].sup.+ ##STR00385## .sup.1H-NMR (400 MHz, CHCl.sub.3-d): .delta.
ppm 0.87-1.01 (m, 10 H) 1.08 (d, J = 6.78 Hz, 3 H) 1.27-1.63 (m, 12
H) 1.66-1.74 (m, 1 H) 1.76 (s, 3 H) 1.97-2.10 (m, 3 H) 2.35-2.57
(m, 5 H) 2.58-2.65 (m, 1 H) 2.65- 2.71 (m, 1 H) 2.77 (td, J = 5.93,
2.32 Hz, 1 H) 2.89-3.05 (m, 4 H) 3.07- 3.34 (m, 8 H) 3.50-3.68 (m,
5 H) 3.72-3.88 (m, 1 H) 4.88-5.09 (m, 1 H) 5.18 (d, J = 10.67 Hz, 1
H) 5.50-5.84 (m, 3 H) 6.01-6.13 (m, 1 H) 6.19-6.36 (m, 1 H). LC/MS
(ESI, m/z), 649.7 [M + H].sup.+ ##STR00386## .sup.1H NMR (400 MHz,
METHANOL-d4) .delta. ppm 0.76- 0.87 (m, 9H), 1.03-1.09 (m, 3H),
1.10-1.13 (m, 3H), 1.13-1.19 (m, 1H), 1.20-1.27 (m, 3H), 1.29- 1.48
(m, 6H), 1.51-1.59 (m, 1H), 1.65-1.71 (m, 3H), 1.73-1.81 (m, 1H),
2.20 (s, 3H), 2.27-2.34 (m, 4H), 2.34-2.44 (m, 2H), 2.44- 2.53 (m,
1H), 2.53-2.59 (m, 1H), 2.76-2.83 (m, 1H), 3.23-3.26 (m, 1H),
3.34-3.52 (m, 7H), 3.67- 3.75 (m, 1H), 4.86-4.92 (m, 1H), 4.92-4.99
(m, 1H), 5.41-5.51 (m, 1H), 5.60-5.71 (m, 1H), 5.72- 5.82 (m, 1H),
6.00-6.07 (m, 1H), 6.37-6.48 (m, 1H). LC/MS (ESI, m/z): 665.73 [M +
H].sup.+ ##STR00387## .sup.1H NMR (400 MHz, CHCl.sub.3-d) .delta.
ppm 0.84-1.01 (m, 11 H) 1.08 (d, J = 6.78 Hz, 3 H) 1.29-1.64 (m, 10
H) 1.63-1.73 (m, 1 H) 1.76 (s, 3 H) 1.94-2.12 (m, 4 H) 2.37-2.58
(m, 4 H) 2.59-2.65 (m, 1 H) 2.68 (dd, J = 7.40, 2.26 Hz, 1 H) 2.77
(td, J = 5.93, 2.32 Hz, 1 H) 3.01-3.30 (m, 4 H) 3.49 (s, 1 H)
3.54-3.67 (m, 2 H) 3.69-3.92 (m, 5 H) 4.13- 4.78 (m, 11 H)
5.13-5.24 (m, 2 H) 5.48-5.61 (m, 1 H) 5.62-5.74 (m, 2 H) 6.04-6.13
(m, 1 H) 6.18- 6.32 (m, 1 H) LC/MS (ESI, m/z), 649.6 [M + H].sup.+
##STR00388## .sup.1H-NMR (400 MHz, MeOH-d4): .delta. ppm 0.88-0.99
(m, 9 H) 1.10 (d, J = 6.78 Hz, 3 H) 1.24 (s, 4 H) 1.42- 1.69 (m, 8
H) 1.77 (d, J = 0.88 Hz, 3 H) 2.43-2.63 (m, 4 H) 2.64- 2.70 (m, 1
H) 2.71-2.82 (m, 5 H) 3.34 (br s, 3 H) 3.37 (s, 2 H) 3.42- 3.57 (m,
5 H) 3.79-3.89 (m, 1 H) 5.06 (s, 2 H) 5.54-5.63 (m, 1 H) 5.64-5.80
(m, 2 H) 6.07-6.16 (m, 1 H) 6.29-6.40 (m, 1 H) LC/MS (ESI, m/z),
621.6 [M + H].sup.+ ##STR00389## .sup.1H-NMR (400 MHz, MeOH-d4):
.delta. ppm 0.88-1.00 (m, 9 H) 1.10 (d, J = 6.65 Hz, 3 H) 1.16-1.27
(m, 4 H) 1.40-1.70 (m, 8 H) 1.77 (d, J = 0.88 Hz, 3 H) 2.28-2.36
(m, 3 H) 2.42 (br t, J = 5.08 Hz, 3 H) 2.47- 2.61 (m, 4 H) 2.68
(dd, J = 8.22, 2.20 Hz, 1 H) 2.74 (td, J = 5.99, 2.20 Hz, 1 H)
3.13-3.17 (m, 1 H) 3.34-3.38 (m, 3 H) 3.47-3.58 (m, 5 H) 3.81-3.87
(m, 1 H) 5.01- 5.10 (m, 2 H) 5.54-5.81 (m, 3 H) 6.06-6.15 (m, 1 H)
6.34 (dd, J = 15.00, 10.98 Hz, 1 H) LC/MS (ESI, m/z), 635.8 [M + H]
##STR00390## .sup.1H-NMR (400 MHz, MeOH-d4): .delta. ppm 0.92 (br
d, J = 6.53 Hz, 9 H), 1.25 (br s, 4 H), 1.36 (s, 3 H), 1.41- 1.74
(m, 6 H), 1.45-1.59 (m, 1 H), 1.64-1.72 (m, 1 H), 1.80 (s, 3 H),
1.85-1.94 (m, 1 H), 2.42 (s, 3 H), 2.50-2.63 (m, 3 H), 2.65-2.73
(m, 1 H), 2.73-2.80 (m, 2 H), 2.88-3.01 (m, 4 H), 3.35-3.38 (m, 3
H), 3.35- 3.40 (m, 3 H), 3.41-3.48 (m, 2 H), 3.52-3.57 (m, 1 H),
3.78-3.89 (m, 1 H), 5.00-5.13 (m, 2 H), 5.53-5.64 (m, 1 H),
5.71-5.82 (m, 1 H), 5.84- 5.94 (m, 1 H), 6.12-6.19 (m, 1 H),
6.49-6.61 (m, 1 H) LC/MS (ESI, m/z), 639.7 [M + H].sup.+
##STR00391## LC/MS (ESI, m/z), 653.79 [M + H].sup.+ ##STR00392##
.sup.1H-NMR (400 MHz, DMSO-d6): .delta. ppm 0.71-0.77 (m, 9 H),
1.03 (s, 5 H), 1.14-1.20 (m, 6 H), 1.23-1.33 (m, 5 H), 1.36-1.48
(m, 5 H), 1.63 (s, 3 H), 1.67-1.75 (m, 1 H), 2.13- 2.24 (m, 4 H),
2.58-2.64 (m, 1 H), 2.68-2.81 (m, 2 H), 3.20-3.20 (m, 3 H),
3.61-3.77 (m, 4 H), 4.28-4.39 (m, 1 H), 4.44-4.53 (m, 1 H), 4.79-
4.88 (m, 2 H), 5.28-5.43 (m, 1 H), 5.52-5.66 (m, 1 H), 5.73-5.85
(m, 1 H), 5.94-6.04 (m, 1 H), 6.27-6.41 (m, 1 H), 6.43-6.53 (m, 2
H), 8.19- 8.29 (m, 1 H) LC/MS (ESI, m/z), 709.5 [M + H].sup.+
##STR00393## .sup.1H NMR (400 MHz, DMSO-d6) .delta. ppm 0.75 (s,
9H), 1.03 (s, 3H), 1.16 (s, 3H), 1.16-1.17 (m, 1H), 1.23-1.33 (m,
4H), 1.36-1.47 (m, 2H), 1.53-1.60 (m, 2H), 1.63 (s, 3H), 1.67-1.75
(m, 1H), 2.10- 2.35 (m, 10H), 2.47-2.53 (m, 2H), 2.65-2.73 (m, 1H),
3.15 (s, 3H), 3.27-3.32 (m, 4H), 3.58-3.68 (m, 1H), 4.25-4.39 (m,
1H), 4.45- 4.52 (m, 1H), 4.69-4.78 (m, 1H), 4.80-4.88 (m, 2H),
5.29-5.42 (m, 1H), 5.53-5.66 (m, 1H), 5.72- 5.84 (m, 1H), 5.93-6.05
(m, 1H), 6.27-6.41 (m, 1H), 8.38-8.46 (m, 1H) LC/MS (ESI, m/z),
723.43 [M + H].sup.+
Compounds 197-200 were synthesized according to Scheme 60.
##STR00394##
Step 1
[0911] To a solution of tri-TES Pladienolide D (1.0 equiv.) in
1,2-dichloroethane (0.2 M) at 20.degree. C. was added DMAP (1.5
equiv.), triethylamine (30 equiv.) and 4-nitrophenyl chloroformate
(10 equiv.). The reaction mixture was stirred at 40.degree. C. for
4 days, and then for 2 h at 60.degree. C. The reaction mixture was
diluted with EtOAc and washed with water, then the layers were
separated. The aqueous layer was extracted with EtOAc (2.times.).
The combined organic extracts were successively washed with water
and brine, dried over MgSO.sub.4, filtered and concentrated in
vacuo. Flash column chromatography (EtOAc in Hexane; silica gel)
afforde the intermediate carbonate. To a mixture of the
intermediate carbonate (1.0 equiv.) in DCM (0.2 M) were added
triethylamine (3.0 equiv.) and amine (2.0 equiv.) and the resulting
mixture was stirred at RT for 1 hour. The reaction mixture was then
concentrated and chromatographed (DCM/MeOH; silica gel) to afford
the carbamate intermediate as a mixture of regio-isomers.
Step 2
[0912] The regio-isomeric mixture of carbamate intermediate (1.0
equiv.) was dissolved in DCM (0.04 M). Hunig's base (124 equiv.)
was added and the reaction mixture was cooled to -78.degree. C. and
hydrogen fluoride pyridine (30 equiv.) was added dropwise before
warming the mixture to rt and stirring overnight at rt. The
reaction mixture was then cooled to -78.degree. C. and saturated
sodium bicarbonate was added dropwise. After addition of sodium
bicarbonate, the mixture was warmed to rt. The organic layer was
isolated and the aqueous layer was extracted with DCM (3.times.).
The combined organic layers were dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuo. The resulting residue
was purified by reverse-phase HPLC purification to afford the each
of desired regio-isomeric products.
TABLE-US-00015 TABLE 12 Compounds 197-200 LCMS data Structure,
Compound #, and Chemical Name .sup.1H NMR data (ES+) ##STR00395##
.sup.1H-NMR (400 MHz, MeOH-d.sub.4): .delta. ppm 0.85- 0.98 (m, 9
H) 1.25 (td, J = 7.40, 4.14 Hz, 1 H) 1.34 (s, 3 H) 1.42-1.69 (m, 9
H) 1.79 (s, 4 H) 1.82-1.96 (m, 2 H) 2.04 (d, J = 9.29 Hz, 3 H) 2.33
(br d, J = 10.04 Hz, 1 H) 2.47-2.54 (m, 2 H) 2.57-2.72 (m, 2 H)
2.87-2.92 (m, 1 H) 3.03 (br s, 2 H) 3.33-3.43 (m, 2 H) 3.43-3.57
(m, 2 H) 3.77-3.84 (m, 1 H) 3.86-3.94 (m, 1 H) 4.50 (br s, 1 H)
4.97-5.11 (m, 2 H) 5.60-5.68 (m, 1 H) 5.73-5.82 (m, 1 H) 5.88 (d, J
= 15.31 Hz, 1 H) 6.15 (br d, J = 11.04 Hz, 1 H) 6.53 (dd, J =
15.25, 10.98 Hz, 1 H) 8.54 (s, 1 H) LC/MS (ESI, m/z), 677.6 [M +
H].sup.+ ##STR00396## .sup.1H-NMR (400 MHz, CHCl.sub.3-d): .delta.
ppm LC/MS (ESI, m/z), 677 [M + H].sup.+ ##STR00397## .sup.1H-NMR
(400 MHz, MeOH-d.sub.4): .delta. ppm 0.88- 0.98 (m, 9 H) 1.26 (td,
J = 7.40, 4.27 Hz, 1 H) 1.32-1.39 (m, 3 H) 1.44- 1.70 (m, 9 H) 1.80
(s, 3 H) 1.83-1.92 (m, 1 H) 2.05 (s, 3 H) 2.32-2.46 (m, 1 H)
2.47-2.70 (m, 11 H) 2.91 (td, J = 5.77, 2.26 Hz, 1 H) 3.43-3.61 (m,
5 H) 3.69 (t, J = 5.83 Hz, 2 H) 3.81 (br dd, J = 9.79, 3.39 Hz, 1
H) 5.00- 5.11 (m, 2 H) 5.64 (dd, J = 15.18, 9.66 Hz, 1 H) 5.73-5.81
(m, 1 H) 5.86- 5.94 (m, 1 H) 6.15 (br d, J = 10.29 Hz, 1 H) 6.54
(dd, J = 15.25, 10.98 Hz, 1 H) LC/MS (ESI, m/z), 709.7 [M +
H].sup.+ ##STR00398## .sup.1H-NMR (400 MHz, CHCl.sub.3-d): .delta.
ppm LC/MS (ESI, m/z), 709.8 [M + H].sup.+
EXAMPLE 201
##STR00399##
[0914] To a mixture of (2 S,3 S,6 S,7R,10R,E)-6-acetoxy
-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan--
2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-dimethyl-12-oxooxacyclo-
dodec-4-en-7-yl piperazine-1-carboxylate (230 mg, .346 mmol;
Example 187) in DCM (8 mL) was added sodium triacetoxyborohydride
(4 equiv.) and then formaldehyde (104 mg, 3.459 mmol) as an aqueous
solution. The mixture was stirred for 20 minutes at rt. After
stirring, the mixture was diluted with methanol and then
concentrated in vacuo onto silica and purifed by silica gel
chromatography (0-15% MeOH/DCM) and concentrated in vacuo to afford
(2 S,3S,6
S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R-
,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-di-
methyl-12-oxooxacyclododec-4-en-7-yl
4-methylpiperazine-1-carboxylate (160 mg, 0.236 mmol, 68.1% yield)
as a colorless oil. [0915] LCMS (ESI, m/z), [M+H].sup.+679.2.
EXAMPLE 202
##STR00400##
[0917] To a mixture of (2 S,3S,6
S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((2R-
,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7-di-
methyl-12-oxooxacyclododec-4-en-6-yl piperazine-1-carboxylate (55
mg, 0.083 mmol; Example 186) in DCM (3 mL) was added
(9H-fluoren-9-yl)methyl (2-oxoethyl)carbamate (46.5 mg, 0.165 mmol)
and sodium triacetoxyborohydride (52.6 mg, 0.248 mmol). The mixture
was stirred at rt for 20 minutes and then concentrated in vacuo.
The resulting residue was purified by silica gel column
chromatography (0-10% MeOH/DCM) and concentrated in vacuo. The
isolated material was diluted with DMF (3 mL) and to that mixture
was added diethylamine (121 mg, 1.655 mmol). The mixture was
stirred until it the starting material was consumedat rt before
concentrating in vacuo. The resulting residue was purified via
reverse phase HPLC to afford
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl-4-(2-aminoethyl)piperazine-1--
carboxylate (6 mg, 8.48 umol, 10.25% yield) as a white solid.
.sup.1H NMR (400 MHz, DMSO-d6): .delta. ppm 0.74-0.86 (m, 9H)
1.04-1.15 (m, 1H) 1.23 (s, 3H) 1.26-1.40 (m, 3H) 1.45 (s, 4H)
1.47-1.51 (m, 1H), 1.52-1.63 (m, 1H) 1.69 (s, 3H) 1.73-1.82 (m, 1H)
1.99 (s, 3H) 2.13-2.42 (m, 9H) 2.53-2.65 (m, 4H) 2.72-2.80 (m, 1H),
3.35-3.41 (m, 3H) 3.65-3.76 (m, 1H) 4.36-4.46 (m, 1H) 4.57-4.66 (m,
1H) 4.79-4.85 (m, 1H) 4.87-4.95 (m, 2H) 5.43-5.56 (m, 1H) 5.64-5.77
(m, 1H) 5.80-5.91 (m, 1H) 6.01-6.12 (m, 1H) 6.33-6.49 (m, 1H). LCMS
(ESI, m/z), 708.2 [M+H].sup.+
EXAMPLE 203
##STR00401##
[0919] To a solution of (2S,3S
,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3-((-
2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3,7--
dimethyl-12-oxooxacyclododec-4-en-6-yl piperazine-1-carboxylate (25
mg, 0.038 mmol; Example 186) in acetone (2 mL) was added ethyl
2-bromoacetate (7.54 mg, 0.045 mmol) and potassium carbonate (3
equiv.). The resulting mixture was stirred for 25 minutes before
adding additional bromo acetate (2 equiv.) and stirring at rt for 1
hour. Subsequently, the mixture was diluted with ethyl acetate and
washed with brine. The organic layer was isolated, dried over
sodium sulfate, filtered, and concentrated in vacuo. The resulting
residue was purified by flash column chromatography (0-15%
MeOH/DCM) to provide
(2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3
S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-y-
l)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
4-(2-ethoxy-2-oxoethyl)piperazine-1-carboxylate (12 mg, 0.016 mmol,
42.5% yield) as a colorless oil. LCMS (ESI, m/z),
[M+H].sup.+751.3
EXAMPLES 204 and 205
[0920] Examples 204 and 205 were prepared via the sequence outlined
in Scheme 61.
##STR00402##
[0920] Step 1:
[0921] A mixture of
(2S,3S,6S,7R,10R,E)-7-hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((trie-
thylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxi-
ran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec--
4-en-6-yl acetate (0.626 g, 0.699 mmol), THF (4.58 mL, 55.925
mmol), ethyl vinyl ether (2.69 ml, 27.963 mmol), PPTS (0.044 g,
0.175 mmol) was stirred overnight. Triethylamine (0.8 eq) was added
to the reaction mixture and stirred for several minutes before
extracting with saturated aqueous sodium bicarbonate. The aqueous
layer was isolated and extracted with EtOAc. The combined organic
layers were dried over anhydrous sodium sulfate, filtered, and
contrated in vacuo to afford
(2S,3S,6S,7R,10R,E)-7-(1-ethoxyethoxy)-3,7-dimethyl-2-4R,2E,4E)-6-methyl--
6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan--
2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyc-
lododec-4-en-6-yl acetate as a mixture of diastereomers (636 mg,
0.657 mmol, 94% yield).
[0922] .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. ppm 0.60 (q,
J=7.65 Hz, 19H) 0.78-0.92 (m, 10H) 0.96 (t, J=7.91 Hz, 28H)
1.16-1.30 (m, 8H) 1.32-1.35 (m, 1H) 1.38 (br s, 5H) 1.43-1.62 (m,
7H) 1.71 (d, J=6.65 Hz, 4H) 2.02-2.08 (m, 3H) 2.33-2.53 (m, 3H)
2.53-2.59 (m, 1H) 2.79-2.87 (m, 1H) 3.42-3.67 (m, 2H) 3.68-3.76 (m,
1H) 3.78-3.86 (m, 1H) 4.94-5.13 (m, 2H) 5.14-5.20 (m, 1H) 5.57-5.78
(m, 3H) 6.03-6.13 (m, 1H) 6.35-6.47 (m, 1H).
Step 2:
[0923] A mixture of
(2S,3S,6S,7R,10R,E)-7-(1-ethoxyethoxy)-3,7-dimethyl-2-4R,2E,4E)-6-methyl--
6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan--
2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyc-
lododec-4-en-6-yl acetate (1.4 g, 1.447 mmol), potssium carbonate
(0.300 g, 2.17 mmol), and methanol (14.47 mL, 1.447 mmol) was
stirred for 1 hr. EtOAc and saturated aqueous ammonium chloride
were added to the mixture and the organic layer was isolated. The
aqueous layer was then extraxted three times with EtOAc, and the
organic layers were combined, dried over anhydrous sodium sulfate,
filtered, and concentrated to dryness to afford (4R,7R,8S ,11S
,12S,E)-7-(1-ethoxyethoxy)-8-hydroxy-7,11-dimethyl-12-((R,2E,4E)-6-methyl-
-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-
-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-4-((triethylsilyl)oxy)oxacyclododec-
-9-en-2-one (1 g, 1.080 mmol, 74.7% yield).
Step 3:
[0924]
(4R,7R,8S,11S,12S,E)-7-(1-ethoxyethoxy)-8-hydroxy-7,11-dimethyl-12--
((R,2E,4E)-6-methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triet-
hylsilyl)oxy)pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-4-((triethylsily-
l)oxy)oxacyclododec-9-en-2-one (1 g, 1.08 mmol), DCM (0.1 M),
Hunig's Base (5.0 equiv.), DMAP (1.0 equiv.), and 4-nitrophenyl
chloroformate (1.8 equiv.) were combined and stirred overnight.
Aqueous sodium hydroxide (1N) was added to the resulting mixture
and the organic layer was isolated. The aqeous layer was then
extracted three times with DCM. The organic layers were combined,
dried over anhydrous sodium sulfate, filtered, and concentrated in
vacuo. The resulting residue was diluted with DCM (0.1 M), and to
that mixture was added Hunig's Base (5.0 equiv.) and amine (3.0
equiv.), followed by stirring for 2 hours. The resulting mixture
was then purified by silica gel chromatography (1-10% MeOH in DCM)
to afford carbamate intermediate.
[0925] Carbamate Intermediate #1:
##STR00403##
[0926]
(2S,3S,6S,7R,10R,E)-7-(1-ethoxyethoxy)-3,7-dimethyl-2-((R,2E,4E)-6--
methyl-6-((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)-
pentan-2-yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy-
)oxacyclododec-4-en-6-yl 4-methylpiperazine-1-carboxylate (700 mg,
0.666 mmol, 61.6% yield). LCMS (ESI, m/z), 1052.6 (M+H).sup.+
[0927] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 0.60-0.70 (m,
18H) 0.82-1.03 (m, 38H) 1.12-1.26 (m, 5H) 1.26-1.36 (m, 6H) 1.43
(s, 3H) 1.45-1.64 (m, 7H) 1.77 (s, 4H) 1.88-1.99 (m, 1H) 2.30 (s,
3H) 2.36-2.46 (m, 5H) 2.46-2.65 (m, 3H) 2.82-2.93 (m, 1H) 3.55 (s,
6H) 3.71-3.81 (m, 1H) 3.84-3.98 (m, 1H) 4.88-5.00 (m, 2H) 5.09-5.18
(m, 1H) 5.52-5.63 (m, 1H) 5.72-5.88 (m, 2H) 6.09-6.19 (m, 1H)
6.45-6.57 (m,1H).
[0928] Carbamate Intermediate #2
##STR00404##
[0929] (2S,3S
,6S,7R,10R,E)-7-(1-ethoxyethoxy)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((t-
riethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)-
oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclodod-
ec-4-en-6-yl piperazine-1-carboxylate (330 mg, 0.318 mmol, 69.4%
yield).
Step 4:
[0930] Tert-butanol (0.16 M), THF (0.08 M), and PPTS (3.0 equiv.)
were combined and stirred at RT. (2S,3S,6S
,7R,10R,E)-7-(1-ethoxyethoxy)-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((trie-
thylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxi-
ran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec--
4-en-6-yl 4-methylpiperazine-1-carboxylate (1.0 equiv.) was added
to the mixture and it was stirred overnight. Subsequently,
saturated brine was added and the mixture was stirred for 30
minutes. The organic layer was isolated and the aqueous layer was
extracted three times with DCM. The organic layers were combined,
dried over anhydrous sodium sulfate, filtered, concentrated in
vacuo, and the resulting residue was purified by silica gel
chromatography (0-100% EtOAc in Hexane) to afford tri-TES protected
intermediate.
[0931] Tri-TES Protected Intermediate #1:
##STR00405##
[0932]
(2S,3S,6S,7R,10R,E)-7-hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6--
((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2--
yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclo-
dodec-4-en-6-yl 4-methylpiperazine-1-carboxylate (172 mg, 0.176
mmol, 41.1% yield) LCMS (ESI, m/z), 980.144 (M+H).sup.+
[0933] Tri-TES Protected Intermediate #2:
##STR00406##
[0934]
(2S,3S,6S,7R,10R,E)-7-hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6--
((triethylsilyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2--
yl)oxiran-2-yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclo-
dodec-4-en-6-yl piperazine-1-carboxylate (0.49 g, 96%) LCMS (ESI,
m/z), 966.1 (M+H).sup.+.
[0935] Step 5:
[0936] (2S,3S
,6S,7R,10R,E)-7-hydroxy-3,7-dimethyl-2-((R,2E,4E)-6-methyl-6-((triethylsi-
lyl)oxy)-7-((2S,3S)-3-((2S,3S)-3-((triethylsilyl)oxy)pentan-2-yl)oxiran-2--
yl)hepta-2,4-dien-2-yl)-12-oxo-10-((triethylsilyl)oxy)oxacyclododec-4-en-6-
-yl 4-methylpiperazine-1-carboxylate (100 mg, 0.102 mmol), DCM (371
equiv.), and DIPEA (191 equiv.) were combined and cooled to
-78.degree. C. Hydrogen fluoride-pyridine (30 equiv) was added and
the mixture was warmed to RT and stirred overnight. The mixture was
then cooled in an icebath, and then saturated aqueous sodium
bicarbonate was added. The resulting mixture was extracted with DCM
and the organic layers were combined, dried over anhydrous sodium
sulfate, filtered, concentrated in vacuo, and chromatographed on
silica gel (MeOH/DCM) to afford the desired compound.
EXAMPLE 204
##STR00407##
[0938]
(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
4-methylpiperazine-1-carboxylate (31.6 mg, 0.050 mmol, 48.6%
yield)
[0939] MS (ESI, m/z), 637.6 (M+H).sup.+
[0940] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 0.86-0.98 (m, 9H)
1.20-1.23 (m, 3H) 1.23-1.32 (m, 2H), 1.34 (s, 3H) 1.35-1.70 (m, 7H)
1.78 (d, J=0.75 Hz, 3H) 1.83-1.93 (m, 1H) 2.30 (s, 3H) 2.41 (br t,
J=4.77, Hz, 4H) 2.52 (dd, J=3.39, 1.63 Hz, 3H) 2.65-2.72 (m, 1H)
2.86-2.95 (m, 1H) 3.38-3.73 (m, 5H) 3.76 -3.88 (m, 1H) 4.95 (s, 1H)
5.03-5.13 (m, 1H) 5.51-5.63 (m, 1H) 5.66-5.78 (m, 1H) 5.82-5.93 (m,
1H), 6.08-6.20 (m, 1H) 6.48-6.61 (m, 1H).
EXAMPLE 205
##STR00408##
[0942]
(2S,3S,6S,7R,10R,E)-7,10-dihydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R-
)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl-
)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl
piperazine-1-carboxylate (50 mg, 78% yield)
[0943] MS (ESI, m/z), 623.7 (M+H).sup.+
[0944] .sup.1H NMR (400 MHz, MeOH-d4) .delta. ppm 0.88-0.99 (m, 9H)
0.98-1.05 (m, 2H) 1.25 (s, 4H) 1.21-1.27 (m, 1H) 1.34-1.37 (m, 4H)
1.48-1.73 (m, 5H) 1.76-1.83 (m, 3H) 1.85-1.93 (m, 1H) 2.46-2.74 (m,
4H) 2.88-2.95 (m, 1H) 3.21 (s, 4H) 3.51-3.60 (m, 1H) 3.78 (s, 5H)
4.94-5.01 (m, 1H) 5.05-5.11 (m, 1H) 5.56-5.66 (m, 1H) 5.70-5.79 (m,
1H) 5.86-5.93 (m, 1H) 6.08-6.24 (m, 1H) 6.45-6.63 (m, 1H)
[0945] Biological Assays
[0946] Cell Viability Assay Protocol
[0947] Cells (WiDr and Panc05.04 obtained from ATCC) were seeded in
96-well plates, with 2000 cells/100 .mu.L/well, and incubated
overnight. Spent media was removed, and fresh media containing 9
different concentrations of compound (100 .mu.L/well) were added,
with DMSO concentration from compound stock solution adjusted to be
0.1%. Each compound treatment was done in duplicate or triplicate
at each concentration.
[0948] Another plate with cells seeded was dedicated as a time zero
(Tz) plate, to which was added 0.1% DMSO in media (100 .mu.L/well)
followed by CellTiter-Glo.RTM. reagent (Promega Corporation,
Madison, Wis.) (50 .mu.L/well) for ATP measurement as a surrogate
of cell viability. Average value from measurement of multiple wells
of this plate is used as Tz. Compound-treated plates were incubated
for 72 hr at 37.degree. C. Then, CellTiter-Glo.RTM. reagent (50
.mu.L/well) was added and ATP was measured. Average value from
measurement of the duplicate or triplicate compound-treated wells
is used as Ti, and seeded plates with medium having 0.1% DMSO
without compound is used as control growth (C).
[0949] Percentage growth inhibition/Percentage viability was
calculated as:
[0950] [(Ti-Tz)/(C-Tz)].times.100 for concentrations for which
Ti>/=Tz
[0951] [(Ti-Tz)/Tz].times.100 for concentrations for which
Ti<Tz. [0952] *time zero (Tz), control growth (C), and test
growth in the presence of compound (Ti) Percentage growth
inhibition/Percentage viability are plotted versus compound
concentration to determine E.sub.max.
[0953] Growth inhibition of 50% (GI.sub.50) was calculated from
[(Ti-Tz)/(C-Tz)].times.100=50, which is the drug concentration
resulting in a 50% reduction in the net increase of ATP in control
growth (C) during the compound treatment.
[0954] In Vitro Splicing (biochemical) Assay Protocol
[0955] Biotin-labeled pre-mRNA of an adenovirus type 2 construct
with a deletion of intervening sequence (Ad2) (Berg, M. G., et al.
2012 Mol. Cell Bio., 32(7):1271-83) was prepared by in vitro
transcription. The Ad2 construct containing Exon 1 (41
nucleotides), Intron (231 nucleotides), and Exon 2 (72 nucleotides)
was generated by gene synthesis and cloned into the EcoRI and XbaI
sites of pGEM.RTM.-3Z vector (Promega) by Genewiz.RTM. (South
Plainfield, N.J). The plasmid was then linearized by XbaI digestion
and purified. In vitro transcription and purification of
transcribed pre-mRNA were performed using the MEGAscript.RTM. T7
transcription kit (Invitrogen.TM., Life Technologies.TM., Grand
Island, N.Y.) and MEGAclear.TM. transcription clean-up kit
(Invitrogen.TM., Life Technologies.TM., Grand Island, N.Y.),
respectively, following the manufacturer's instructions. The ratio
of biotin-16-UTP (Roche Diagnostics Corporation, Indianapolis,
Ind.) to cold UTP was 1:13 to incorporate approximately two biotin
molecules per spliced Ad2 mRNA.
[0956] In vitro splicing assay was performed at 30.degree. C. in 25
.mu.L reaction mixtures containing 95 .mu.g HeLa nuclear extract
(Promega Corporation, Madison, Wis.), 47 nM Ad2 pre-mRNA, 25U
RNasin RNase inhibitor (Promega Corporation, Madison, Wis.),
1.times. SP buffer (0.5 mM ATP, 20 mM creatine phosphate, 1.6 mM
MgCl.sub.2), and compounds in DMSO (with 1% final concentration of
DMSO). After 90 min of incubation, the reaction was stopped by
addition of 18 .mu.L of 5M NaCl, and the mixtures were incubated
with 10 .mu.L of M-280 streptavidin-coated magnetic beads
(Invitrogen.TM., Life Technologies, Grand Island, N.Y.) for 30 min
at room temperature to capture Ad2 pre- and spliced mRNA. The beads
were washed twice with 100 uL buffer containing 10 mM Tris pH=7.5,
1mM EDTA and 2M NaCl, and then incubated in RNA gel loading buffer
containing 95% formamide at 70.degree. C. for 10 min to elute the
RNAs. Ad2 RNAs were resolved by 6% TBE-UREA gel, transferred to a
nylon membrane, UV cross-linked, and probed with an IRDye.RTM.
labeled streptavidin (LI-COR, Lincoln, Nebr.). The amount of
spliced RNA was quantified by measuring the band fluorescent
intensity using LI-COR Image Studio software.
[0957] Results
[0958] Data are reported in Table 13 below. E.sub.max refers to the
maximum achievable response to a compound in a tested dose range,
with a negative value indicating cellular lethality. A larger
negative E.sub.max value indicates greater cellular lethality for a
particular compound. For example, in Panc 05.04 cells, a mutant
SF3B1 cell line, the larger negative Emax value indicates that
Compound 1 had greater cellular lethality than Compound 7.
[0959] WiDr-R cells are colon cancer cells which have a
chemically-induced R1074H mutation and have been shown to be
resistant to pladienolide B in terms of growth inhibition (Yokoi,
A., et al., 2011 FEBS Journal, 278:4870-4880). The
counter-screening of compounds in this viability assay with a
"resistant" WiDr-R cell line may indicate whether these compounds
have off-target effect(s). Compounds that lack growth inhibitory
(GI.sub.50) activity in the resistant WiDr-R cell line but maintain
activity in the parental WiDr cell line suggests that on-mechanism
splicing modulation is responsible for the growth inhibition which
is observed in the parental WiDr cell line.
[0960] Scintillation Proximity Assay (SPA) with [.sup.3H]-labelled
Pladienolide Probe
[0961] Batch immobilization of anti-SF3B1 antibody (MBL) to
anti-mouse PVT SPA scintillation beads (PerkinElmer) was prepared
as follows: for every 2.5 mg of nuclear extracts, 5 .mu.g
anti-SF3B1 antibody and 1.5 mg of beads were mixed in 150 .mu.l
PBS. The antibody-bead mixture was incubated for 30 min at RT and
centrifuged at 18,000 g for 5 min. 150 p1 PBS was used to resuspend
every 1.5 mg antibody-bead mixture. The beads were suspended and
added to the prepared nuclear extracts. The slurry was incubated
for 2 h at 4.degree. C. with gentle mixing. The beads were then
collected by centrifuging at 18,000 g for 5 min, and washed twice
with PBS+0.1% Triton X-100. After a final centrifugation step,
every 1.5 mg of beads was suspended with 150 .mu.l of PBS. The SF3b
complexes were tested for [.sup.3H]-labelled pladienolide probe
binding ([.sup.3H]-PB), synthesized as previously described (Kotake
et al., 2007). 100 .mu.L binding reactions were prepared with 50
.mu.l bead slurry and by adding varying concentrations of PB or
PB--OH, and after 30 min pre-incubation, 2.5 nM [.sup.3H]--PB was
added. The mixture was incubated for 30 min, and luminescence
signals were read using a MicroBeta2 Plate Counter (PerkinElmer).
Prism 6 (Graphpad) was used for non-linear regression curve fitting
of the data.
[0962] Key for Table 13: [0963] WiDr cells=Colon cancer cells;
wildtype SF3B1 [0964] WiDr-R cells=Colon cancer cells;
chemically-induced SF3B1 mutant which is resistant to E7107 (R1074H
mutation) Panc 05.04 cells=Pancreatic cancer cells; Q699H and K700E
mutations in SF3B1 [0965] SPA=Scintillation proximity assay
TABLE-US-00016 [0965] TABLE 13 Biological Activity of Example
Compounds Panc Panc 05.04 05.04 SPA (mt (mt (wt SF3B1 SF3B1 WiDr-
SF3B1 cells) cells), WiDr R cells) E.sub.max GI.sub.50 GI.sub.50
GI.sub.50 IC.sub.50 Structure and Compound # (%) (nM) (nM) (nM)
(nM) ##STR00409## -87.275 27.349 26.401 >1000 4.355 ##STR00410##
-92.825 326.677 554.751 >10000 53.368 ##STR00411## 40.550
4589.385 1941.512 >10000 472.770 ##STR00412## -91.760 42.549
39.130 >1000 6.215 ##STR00413## -94.128 20.087 4.826 >1000
2.746 ##STR00414## -94.410 250.128 172.085 >10000 ##STR00415##
-91.152 74.414 46.727 >10000 17.965 ##STR00416## -92.151 25.017
21.224 >10000 2.397 ##STR00417## -87.370 54.563 32.637 >10000
4.853 ##STR00418## 198.03 86.457 >10000 ##STR00419## 587.41
307.763 >10000 ##STR00420## 40.097 2090.471 8131.147 >10000
##STR00421## -90.898 76.514 71.613 >1000 60.673 ##STR00422##
-96.801 64.282 246.764 >1000 ##STR00423## -91.832 44.669 137.681
>10000 10.740 ##STR00424## -89.983 52.598 38.111 >10000
27.299 ##STR00425## -79.344 2.110 0.983 >1000 2.721 ##STR00426##
-74.649 82.242 49.693 >10000 15.049 ##STR00427## -83.432 35.134
24.651 >10000 80.359 ##STR00428## -89.552 45.075 51.472
>10000 96.546 ##STR00429## 15.45 91.252 >10000 ##STR00430##
-88.833 203.085 105.995 >1000 235.639 ##STR00431## 67.97 62.92
>1000 ##STR00432## -90.269 3.051 3.832 >1000 2.103
##STR00433## -94.750 88.064 89.925 >1000 ##STR00434## -77.223
20.340 29.407 >1000 14.018 ##STR00435## -56.00 35.40 33.788
>1000 ##STR00436## -92.84 30.49 36.628 >1000 ##STR00437##
-85.416 10.054 14.697 >1000 ##STR00438## -93.737 7.817 9.605
>1000 3.729 ##STR00439## -68.065 1.123 1.035 >1000 2.772
##STR00440## -97.033 16.654 19.777 >1000 ##STR00441## -90.19
4.86 2.844 >1000 ##STR00442## -82.07 2.25 1.697 >1000
##STR00443## 90.700 31.462 47.213 >1000 9.557 ##STR00444## 1.960
545.544 204.979 >10000 234.867 ##STR00445## -16.082 537.655
230.686 >10000 45.128 ##STR00446## -28.235 408.252 343.407
>10000 ##STR00447## 6.969 1288.909 252.246 >1000 ##STR00448##
-49.209 53.638 34.307 >1000 5.693 ##STR00449## -90.908 59.215
18.672 >1000 9.201 ##STR00450## -22.224 155.424 60.103 >1000
38.444 ##STR00451## -90.107 26.610 31.754 >1000 6.373
##STR00452## -78.056 65.979 76.010 >1000 13.057 ##STR00453##
-89.491 27.891 19.587 >1000 8.756 ##STR00454## -87.701 37.193
53.113 >1000 8.560 ##STR00455## -41.858 114.631 129.855 >1000
71.254 ##STR00456## -60.371 126.216 132.822 >1000 ##STR00457##
-83.573 38.919 36.709 >1000 7.230 ##STR00458## -60.16 25.08
16.250 >1000 ##STR00459## 35.309 2545.788 >1000.000 >1000
479.605 ##STR00460## -29.357 56.386 41.521 >1000 12.258
##STR00461## -95.714 64.987 51.978 >1000 ##STR00462## -23.122
84.867 33.109 >1000 6.869 ##STR00463## -94.348 21.406 21.202
>1000 3.325 ##STR00464## -76.515 39.538 15.971 >1000 26.868
##STR00465## -12.670 103.826 74.907 >1000 7.317 ##STR00466##
35.115 1389.178 1145.992 >10000 ##STR00467## -93.786 13.556
7.178 >1000 2.347 ##STR00468## -90.314 76.078 47.340
##STR00469## -86.908 3.254 12.669 >1000 5.114 ##STR00470##
-92.429 278.544 43.378 >1000 ##STR00471## -87.256 1.658 2.545
>1000 2.825 ##STR00472## -84.513 5.509 5.562 >1000 7.661
##STR00473## -70.370 2.394 2.735 40.140 7.033 ##STR00474## 80.11
77.26 >1000 ##STR00475## 2.52 1.424 21.764 ##STR00476## -86.642
1.198 3.934 >1000 ##STR00477## -93.599 2.593 6.204 >1000
##STR00478## -92.870 40.226 32.782 >1000 ##STR00479## -89.146
11.933 29.094 >1000 16.761 ##STR00480## -68.88 4.52 2.392 14.819
##STR00481## -92.218 22.502 7.641 >1000 ##STR00482## -79.385
1.764 4.297 >1000 1.929 ##STR00483## -92.980 10.383 28.938
>1000 ##STR00484## -79.728 4.434 3.462 221.390 5.142
##STR00485## -92.417 86.299 41.399 1879.784 ##STR00486## 35.028
2635.026 >1000.000 >1000 36.054 ##STR00487## -93.610 2.845
4.774 449.450 ##STR00488## -76.42 7.98 6.393 602.820 ##STR00489##
-93.540 11.681 11.789 861.860 11.552 ##STR00490## -24.659 302.087
137.820 >1000 ##STR00491## -95.353 74.626 294.340 >1000
##STR00492## -58.256 108.430 691.610 >1000 2.255 ##STR00493##
-85.429 33.049 30.233 3992.400 9.701 ##STR00494## -89.812 3.676
22.230 >1000 3.679 ##STR00495## -72.713 1.745 1.235 >1000
4.234 ##STR00496## -59.56 59.43 28.047 >1000 ##STR00497## -84.18
18.62 8.277 >1000 ##STR00498## -95.447 10.381 19.662 >1000
2.514 ##STR00499## -90.025 71.754 65.037 >1000 5.952
##STR00500## 63.471 >4000.000 148.414 >1000 ##STR00501##
-96.595 147.043 22.977 4310.192 ##STR00502## -93.732 22.057 31.918
>10000 6.375 ##STR00503## -94.489 98.272 40.135 >10000 16.134
##STR00504## -92.260 21.238 15.850 >1000 6.265 ##STR00505##
-92.395 59.861 58.701 >1000 6.791 ##STR00506## -91.588 30.954
45.936 >1000 11.891 ##STR00507## -94.716 22.005 15.089 >1000
3.382 ##STR00508## -26.648 955.710 531.915 >1000 503.624
##STR00509## -92.893 29.505 33.604 >1000 8.103 ##STR00510##
-93.100 34.531 38.061 >1000 6.573 ##STR00511## -76.743 100.995
124.520 >1000 9.698 ##STR00512## -95.264 121.789 320.244
>1000 21.857 ##STR00513## -90.949 10.781 8.393 >1000
##STR00514## -77.841 10.825 8.272 >1000 5.670 ##STR00515##
-86.024 1.364 3.805 >1000 2.961 ##STR00516## -83.342 3.374 4.863
>1000 5.435 ##STR00517## -78.99 4.36 2.656 >1000 ##STR00518##
-77.71 6.76 5.055 >1000 ##STR00519## -79.107 0.339 2.198
>1000 ##STR00520## 28.067 2890.203 420.641 >10000 538.320
##STR00521## -96.287 43.969 33.240 2428.955 10.560 ##STR00522##
-94.255 8.070 15.318 >10000 3.784 ##STR00523## 23.951 1717.435
478.606 >10000 142.021 ##STR00524## -65.323 4.885 2.140 821.510
5.281 ##STR00525## -93.253 170.176 74.629 929.153 ##STR00526##
-90.468 6.738 7.836 2443.625 3.712 ##STR00527## -94.777 155.862
135.293 6382.117 71.129 ##STR00528## -83.689 139.143 154.043
-10000
##STR00529## -87.001 6.480 8.588 2835.605 5.422 ##STR00530## -65.34
7.73 3.440 680.844 ##STR00531## -62.754 2.897 2.519 422.835 3.608
##STR00532## -81.612 35.234 45.539 >1000 40.193 ##STR00533##
-85.578 54.749 65.541 >1000 113.381 ##STR00534## >10000
>10000 >10000 ##STR00535## -40.649 882.501 258.063 3819.588
##STR00536## -93.548 75.129 67.116 >10000 7.896 ##STR00537##
48.162 5794.861 1064.595 7760.310 >1200.000 ##STR00538## 52.01
>10000 4284.737 >10000 ##STR00539## 51.96 >10000 810.247
4077.331 ##STR00540## >1000.000 >10000 ##STR00541## -94.806
474.937 454.567 >10000 ##STR00542## -72.647 40.380 45.671
>1000 ##STR00543## -91.386 2.385 4.925 >1000 1.667
##STR00544## -72.301 2.068 1.742 >1000 2.543 ##STR00545##
-94.561 4.443 11.236 >1000 ##STR00546## -94.92 37.27 31.612
>1000 ##STR00547## -90.189 6.549 15.226 >1000 47.804
##STR00548## -83.721 3.139 7.117 >1000 5.692 ##STR00549##
-75.710 33.924 31.569 >1000 ##STR00550## -95.426 12.251 24.983
>1000 ##STR00551## -90.892 19.831 27.644 >1000 5.244
##STR00552## -85.001 77.295 95.904 >1000 40.798 ##STR00553##
-90.365 54.083 76.622 >1000 7.619 ##STR00554## -94.475 23.520
145.219 >1000 ##STR00555## -94.521 38.566 51.840 >1000 14.147
##STR00556## -95.852 5.109 14.700 >1000 ##STR00557## -96.911
51.275 103.451 >1000 4.542 ##STR00558## -88.360 53.333 72.976
>1000 11.236 ##STR00559## -56.978 136.788 262.578 >1000
##STR00560## 58.44 63.59 >5500 ##STR00561## -90.353 6.672 4.508
>1000 2.153 ##STR00562## 19.145 1179.770 375.699 >10000
267.299 ##STR00563## -93.574 18.961 18.526 >1000 14.419
##STR00564## -90.53 31.10 42.835 >10000 ##STR00565## 47.525
6919.552 4039.541 >10000 326.162 ##STR00566## -95.148 6.627
13.974 >1000 2.744 ##STR00567## -95.570 10.952 13.246 >1000
2.342 ##STR00568## -94.237 20.415 29.403 >1000 2.562
##STR00569## 20.875 ##STR00570## -78.560 2.407 2.000 132.081 5.851
##STR00571## -41.520 353.724 194 >10000 ##STR00572## 13.281
1501.265 657 >10000 ##STR00573## 32.802 3786.286 473 >10000
##STR00574## 12.322 932.044 382 >10000 ##STR00575## -12.662
554.116 282 >10000 ##STR00576## -27.091 841.978 440 >10000
##STR00577## 0.954 971.633 593 >10000 ##STR00578## 20.264
1667.412 911 >10000 ##STR00579## -55.378 313.799 167 >10000
##STR00580## 87.197 >10000 559 >10000 >1200 ##STR00581##
76.538 >6400 610 >10000 ##STR00582## 84.545 >6400 538
>10000 ##STR00583## -70.521 248.761 ##STR00584## -26.377 827.322
##STR00585## -39.524 427.157 ##STR00586## -67.307 320.764
##STR00587## -32.036 508.411 ##STR00588## -81.709 97.681
##STR00589## 11.003 720.201 119.866 ##STR00590## -81.172 45.423
5.891 ##STR00591## 12.455 861.913 ##STR00592## -2.901 94.839 30.921
##STR00593## 49.481 3980.021 >1200 ##STR00594## -73.192 26.834
8.778 ##STR00595## -74.150 23.547 5.921 ##STR00596## -47.060 2.051
2.117 ##STR00597## -75.410 1.759 9.228 ##STR00598## -78.918 0.860
2.085 ##STR00599## 0.990 ##STR00600## 1.521 ##STR00601## 18.251
##STR00602## -64.413 3.485 ##STR00603## 29.188 ##STR00604## 14.557
##STR00605## 16.0 ##STR00606## 24.3
Administration of at least one compound chosen from compounds of
Formula I, compounds of Formula II, compounds of Formula III, and
pharmaceutically acceptable salts of any of the foregoing [0966]
CT26 colon cancer cells (0.25.times.10.sup.6; ATCC Cat. # CRL-2638)
are implanted subcutaneously into the right flank of eight-week old
female Balb/c mice (Envigo) in 100 .mu.L of PVS lacking Matrigel.
CT26 tumors are allowed to grow to an average of .about.100
mm.sup.3 before animals are enrolled into the efficacy study. Each
treatment group contains 12 mice. Mice are treated with at least
one compound chosen from compounds of Formula I, compounds of
Formula II, compounds of Formula III, and pharmaceutically
acceptable salts of any of the foregoing, an anti-CTLA4 antibody,
or a combination thereof, at various doses and via various routes
of administration. The at least one compound chosen from compounds
of Formula I, compounds of Formula II, compounds of Formula III,
and pharmaceutically acceptable salts of any of the foregoing is
formulated in a composition containing 5% ethanol and 95%
methylcellulose solution (0.5% methylcellulose). The anti-CTLA4
antibody is formulated in PBS at pH 7. Tumors are measured 3 times
per week for up to 19 days. Tumor volumes are calculated using the
ellipsoid formula: Tumor Volume=(length.times.width.sup.2)/2.
Sequence CWU 1
1
5819PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 1Ser Pro Thr Leu Pro Pro Arg Ser Leu1
5211PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 2His Pro Ser Ile Lys Arg Gly Leu Ser
Ser Leu1 5 1038PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 3Leu Leu Leu Pro His His Val
Leu1 5410PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 4Arg Thr Ala Pro Gly Val Arg
Pro Pro Phe1 5 1059PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 5Arg Pro Gln Lys Ser Ile
Gln Ala Leu1 569PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 6Ala Pro Ala Pro Pro Pro Leu
Pro Ala1 5710PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 7Arg Pro Arg Pro Ser Phe Pro
Val Ser Leu1 5 10810PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 8Arg Pro Lys His Gly Asp
Gly Phe Ser Leu1 5 1099PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 9Gly Pro Ala Pro Gly Lys Thr Gly Leu1 5109PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 10Glu Ala Ala Arg Lys Gly Asn Ser Leu1 5119PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 11Arg Ile Lys Glu Lys Ile Glu Glu Leu1 5129PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 12Glu Ile Lys Lys Arg Phe Arg Gln Phe1 5139PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 13His Glu Ser Ala Ala Met Ala Glu Thr1 5149PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 14Ala Leu Lys Leu Lys Gln Val Gly Val1 5159PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 15Asp Leu Lys Lys Arg His Ile Thr Phe1 5169PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 16Asp Val Lys Arg Asn Asp Ile Ala Met1 51710PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 17Ile Pro Ser Asp His Ile Leu Thr Pro Ala1 5
10189PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 18Thr Val Phe Ser Thr Ser Ser Leu Lys1
5198PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 19Ile Thr Ser Cys Leu Leu Asn Phe1
5209PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 20Arg Ala Ser Pro Val Arg Gly Gln Leu1
5219PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 21Val Val Arg Lys Pro Val Ile Ala Leu1
5229PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 22Leu Leu Ser Glu Lys Lys Lys Ile Ser1
52310PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 23Ala Pro Ala Ser Lys Pro Arg Pro Arg
Leu1 5 10249PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 24Arg Tyr Gly Gln Leu Ser
Glu Lys Phe1 5259PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 25Val Tyr Ile Ser Asn Val
Ser Lys Leu1 5269PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 26Leu Pro Thr Lys Glu Thr
Pro Ser Phe1 5279PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 27Gly Glu Ala Pro Pro Pro
Pro Pro Ala1 5289PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 28Leu Glu Glu Ile Ser Lys
Gln Glu Ile1 5299PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 29Ile Tyr Asn His Ile Thr
Val Lys Ile1 530160PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic polypeptide" 30Val Asp Leu Glu Pro
Thr Val Ile Gly Glu Leu Thr Ser Val Thr Gln1 5 10 15Val Arg Ser Gln
Gly Ala Gly Thr Gly Gly Leu Ser Trp Gly Gly Ser 20 25 30Ala Gly His
Ser Pro Thr Leu Pro Pro Arg Ser Leu Ser Leu Leu Leu 35 40 45Leu Pro
His His Val Leu Gln Met Lys Phe Ala Leu Ala Leu Thr Ala 50 55 60Ser
Ser Ser Thr Leu Ser Asn Ser Ser Gln Ala Arg Lys Met Leu Pro65 70 75
80Ile Thr Met Pro Glu Gly Thr Thr Pro Leu Ala Arg Arg Ser Leu Thr
85 90 95Ser Cys Trp Thr Glu Phe Ala Ser Trp Leu Thr Ser Ala Pro Val
Phe 100 105 110Arg Ala Ser Trp Phe Ser Thr Ala Leu Val Gly Glu Leu
Val Leu Gly 115 120 125Ser Pro Arg Cys Ser Trp Asn Val Ser Gln Leu
Ile Met Ala Arg Ser 130 135 140Pro Ser Trp Ser Ser Pro Phe Thr Arg
Arg Pro Arg Phe Pro Gln Leu145 150 155 1603116PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 31Ala Pro Pro Arg Ser His Pro Ser Ile Lys Arg Gly Leu Ser
Ser Leu1 5 10 153228PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 32Met Val Arg Arg Ala Arg
Trp Pro Gly Gly Arg Gly Glu Ala Arg Lys1 5 10 15Ala Pro Arg Thr Ala
Pro Gly Val Arg Pro Pro Phe 20 2533199PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 33Trp Val Asn Cys Leu Phe Val Ser Gly Arg Ala Ala Ala
Gly Gly Gly1 5 10 15Gly Gly Gly Ala Val Pro Pro Tyr Leu Glu Leu Ala
Gly Pro Pro Phe 20 25 30Leu Leu Leu Thr Leu Ile Arg Ile Gly Leu Gly
Arg Arg Ser Gly Arg 35 40 45Ala Gly Gly Arg Ala Gly Thr Gln Cys Gly
Gly Glu Arg Gly Pro Gly 50 55 60Phe Ala Ala Phe Arg Pro Leu Arg Pro
Phe Arg Arg Leu Arg Val Cys65 70 75 80Ala Val Cys Val Arg Gly Ser
Ala Leu Gly Arg Ser Val Gly Leu Pro 85 90 95Arg Gly Gly Ala Ala Gly
Ala Pro Phe Ser Ser Ser Pro Ala Pro His 100 105 110Pro Arg Arg Val
Leu Cys Arg Cys Leu Leu Phe Leu Phe Phe Ser Cys 115 120 125His Asp
Arg Arg Gly Asp Ser Gln Pro Tyr Gln Val Pro Ala Glu Ala 130 135
140Gly Val Glu Gly Leu Glu Gly Ala Gly Gly Gly Arg Glu Gly Leu
Leu145 150 155 160Leu Glu Arg Arg Pro Gln Lys Ser Ile Gln Ala Leu
Arg Cys Asn Thr 165 170 175Ser Glu Thr Ser Thr Ala Asp Pro Leu Lys
Ile Pro Gly Leu Val Pro 180 185 190Leu Ala Leu Ser Ser Lys Val
19534101PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic polypeptide" 34Met Pro Leu Pro Val
Gln Val Phe Asn Leu Gln Val Thr Ser Arg Gly1 5 10 15Arg Pro Gly Pro
Pro Arg Pro Arg Ala Pro Arg His Trp Gly Arg Ala 20 25 30Glu Val Glu
Gln Gly Arg Gly Ala Cys Ala Arg Ser Arg Ser Gly Thr 35 40 45Leu Arg
Ala Gly Pro Pro Arg Ala Ala Arg Val Gly Gly Cys Arg Ala 50 55 60Glu
Gly Ala Ser Pro Pro Trp Leu Arg Ala Ala Ile Gly Gly Arg Arg65 70 75
80Ala Ala Pro Ala Pro Pro Pro Leu Pro Ala Ala His Gly Arg Gly Ser
85 90 95Arg Pro Pro Arg Arg 10035162PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 35Gln Pro Ala Gln Pro Arg Thr Gly Ala Pro Ala Arg Arg
Pro Arg Pro1 5 10 15Arg Pro Ser Phe Pro Val Ser Leu Arg Ser Ala Ala
Pro Pro Thr Gly 20 25 30Thr Ala Gly Gly Thr Gly Arg Phe Val Leu Arg
Pro Gly Glu Ser Gly 35 40 45Ala Gly Gly Gly Gly Asp Ala Trp Asp Thr
Gly Leu Gln Ala Arg Arg 50 55 60Gly Thr Ala Ala Gly Thr Ser Gly Ala
Pro Asn Arg Ser Gln Leu Ser65 70 75 80Ser Leu Thr Phe Pro Ala Gln
Leu Arg Arg Ile Gly Val Ser Gly Arg 85 90 95Lys Pro Gly Ala Gly Gly
Arg Leu Gly Pro Gly Ser Arg Thr Cys Ala 100 105 110Pro Arg Cys Leu
Pro Arg Ala Arg Arg Gly Pro Gly Ala His Pro Arg 115 120 125Gly Gly
Arg Cys Pro Pro Ala Glu Thr Ala Leu Phe Arg Glu Ala Glu 130 135
140Glu Gly Thr Gln Lys Tyr Ser Leu Pro Ser Asp Pro Ala Gly Gln
Ala145 150 155 160Ala Phe3639PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 36Phe Arg Leu His Thr Gly Pro Val Ser Pro Val Gly Gly
Arg Arg Gln1 5 10 15Met Gly Arg Pro Lys His Gly Asp Gly Phe Ser Leu
Gln Val Cys Ser 20 25 30Phe Ile Met Glu Gln Asn Gly
353771PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 37Gly Val Val Glu Ile Thr Gly Glu
Pro Pro Cys Ser Cys Arg Gly Glu1 5 10 15Glu Glu Ala Ser Arg Ala Gly
Arg Ala Gly Gly Val Arg Leu Lys Arg 20 25 30Gly Ser Arg Gly Pro Gly
Glu Leu Asn Val Gly Pro Ala Pro Gly Lys 35 40 45Thr Gly Leu Leu Ile
Pro Leu Leu Arg Asn Trp Glu Cys Gly Ser Leu 50 55 60Leu Arg Ala Leu
Ser Ala Leu65 703814PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 38Lys Met Gly Phe Pro Glu
Ala Ala Arg Lys Gly Asn Ser Leu1 5 103917PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 39Leu Glu Ala Arg Ile Lys Glu Lys Ile Glu Glu Leu Gln Gln
Ala Leu1 5 10 15Ile4016PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 40Glu Ile Lys Lys Arg Phe Arg Gln Phe Lys Gln Ala Val Tyr
Lys Gln1 5 10 154116PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 41Ala His Glu Ser Ala Ala
Met Ala Glu Thr Leu Gln His Val Pro Ser1 5 10 154216PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 42Asn Arg Pro Ser Val Gln Ala Ala Leu Lys Leu Lys Gln Val
Gly Val1 5 10 154319PRTArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic peptide" 43Lys Thr Asp Asp Leu Lys
Lys Arg His Ile Thr Phe Thr Leu Gly Cys1 5 10 15Gly Ile
Cys4416PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 44Met Lys Leu Asp Glu Asp Val Lys Arg
Asn Asp Ile Ala Met Ala Ile1 5 10 154526PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 45Asn Ser Ile Ser Gln Ile Pro Ser Asp His Ile Leu Thr Pro
Ala Leu1 5 10 15Phe Ile Thr Phe Met Thr Ile Leu Asp Leu 20
254624PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 46Thr Val Phe Ser Thr Ser Ser Leu Lys
Leu Asn Gln Pro Gln Lys Tyr1 5 10 15Leu Lys Met Lys Ser Trp Pro Cys
204724PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 47Ala Glu Glu Asp Arg Arg Lys Lys Val
Ile Thr Ser Cys Leu Leu Asn1 5 10 15Phe Asn Leu Ser Lys Ala Gln Ser
204834PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 48Arg Ser Phe Ser Thr Ser Ala Gln
Val Gly Gln Thr Arg Gly Gly Leu1 5 10 15Gln Ala Glu Ala Pro Arg Pro
Gly Pro Arg Ala Ser Pro Val Arg Gly 20 25 30Gln
Leu4916PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 49Arg Gly Tyr Val Val Arg Lys Pro Val
Ile Ala Leu Ser Val Lys Ile1 5 10 155086PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 50Val Asp Met Asp Phe Gly Thr Gly Gly Gln Gly Ala Gly
Pro Val Gly1 5 10 15Arg Gly Lys Asp Trp Ser Cys Thr Leu Ala Val His
Leu Leu Ser Glu 20 25 30Lys Lys Lys Ile Ser Phe Ser Gln Ile Asp Arg
Ala Trp Gly Gly Ser 35 40 45Gln Gly Thr Val Leu Asp Lys Trp Gly Pro
Gly Val Val Ser Glu Leu 50 55 60His Pro Ser Ala Lys Glu Val Ser Val
Gly Arg Asn Ser Val Glu Ser65 70 75 80Leu Met Thr Trp Ala Ser
855166PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 51Glu Lys Gly Ser His Glu Glu Glu
Val Arg Val Pro Ala Leu Ser Trp1 5 10 15Gly Arg Pro Arg Ala Pro Ala
Pro Ala Ser Lys Pro Arg Pro Arg Leu 20 25 30Asp Leu Asn Cys Leu Trp
Leu Arg Pro Gln Pro Ile Phe Leu Trp Lys 35 40 45Leu Arg Pro Arg Pro
Val Pro Ala Ala Thr Pro Leu Thr Gly Pro Leu 50 55 60Pro
Leu655217PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 52Arg Tyr Gly Gln Leu Ser
Glu Lys Phe Asn Arg Arg Lys Val Met Asp1 5 10
15Ser5315PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 53Met Val Tyr Ile Ser Asn
Val Ser Lys Leu Cys Phe Ser Lys Met1 5 10 155448PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 54Asn Thr Leu Pro Thr Lys Glu Thr Pro Ser Phe Leu Leu
Asn Pro His1 5 10 15Thr Ser Trp Val Pro Arg Pro His Arg Glu Ala Pro
Arg Leu Arg Val 20 25 30Gly Val Ala Ala Pro Leu Gln Arg Pro Leu Pro
Ala Leu His Ser His 35 40 455558PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 55Phe Gly Asp Ile Tyr Leu Gly Glu Ala Pro Pro Pro Pro
Pro Ala Ala1 5 10 15Arg Arg Pro Gly Pro Cys Gly Cys Gln Asp Gln Ala
Arg Ser Arg Lys 20 25 30Glu Val Val Ala Pro Ala Gly Ser Pro Arg Lys
Ser Arg His Arg Arg 35 40 45Ile Val Ala Arg Thr Gln Arg Pro Leu Gly
50 555617PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 56Gly Ser Ala Ser Asp Leu
Leu Glu Glu Ile Ser Lys Gln Glu Ile Ser1 5 10
15Phe5716PRTArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic peptide" 57Gln Leu Ile Tyr Asn His
Ile Thr Val Lys Ile Asn Leu Gln Gly Asp1 5 10
15584PRTUnknownsource/note="Description of Unknown DEAH box
helicase 9 (DHX9) peptide" 58Asp Glu Ala His1
* * * * *